Method and molecular diagnostic device for detection, analysis and identification of genomic DNA

ABSTRACT

At least one exemplary embodiment of the invention is directed to a molecular diagnostic device that comprises a cartridge configured to eject samples comprising genomic material into a microfluidic chip that comprises an amplification area, a detection area, and a matrix analysis area.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/073,318, filed Mar. 28, 2011, which is a continuation of U.S. patentapplication Ser. No. 11/514,156, filed Sep. 1, 2006, now U.S. Pat. No.7,915,030, which claims the benefit of priority from U.S. ProvisionalPatent Application No. 60/712,813, filed Sep. 1, 2005, the content ofeach application is hereby incorporated by reference herein in itsentirety.

BACKGROUND OF THE INVENTION Field of the Invention

The invention is directed toward a molecular diagnostic device that canbe used to characterize genomic material that may be present in asample.

Related Background Art

In the biotechnological field, there is a need for rapid identificationof organisms, such as bacteria and viruses, in a variety of samples(e.g., environmental and medical). For example, rapid characterizationof genomic material isolated from a bacterium (i.e., identification ofthe species and/or strain of the bacterium) may be necessary to providequality assurance for, e.g., a local water supply, a hospital (includinghospitalized patients therein), or a food processing plant; i.e., it maybe necessary to monitor various samples, including but not limited tosamples of air, dust, water, blood, tissues, plants, foodstuffs, etc.,for the presence of contaminating organisms, and to identify thecontaminating organisms prior to consumption, exposure, and/or use bythe public, or during use by the public, or in tissue or blood samplesobtained from a patient or another member of the public.

Standard microbiological methods for identifying an organism, e.g.,culturing and Gram-staining or testing of other biochemical properties,are imprecise and often cannot differentiate among different organisms,let alone different strains of an organism. More precise methods foridentifying an organism are based on the genomic DNA of the organism.Two such methods of identification are the polymerase chain reaction(PCR), for which technological developments (e.g., automated inline PCRplatforms) have increased its level of throughput and automation, andthe newer method of waveform profiling.

Because PCR exponentially amplifies DNA, it can be used to detect smallamounts of genomic material. However, because PCR requires primers thatare specifically complimentary to sequences of the genomic material thatare known and bracket a DNA locus of interest, PCR is limited in that itcan only be used for the characterization of known organisms. In otherwords, the investigator is required to know or guess the identity of theorganism (i.e., the appropriate pair of primers to use) prior to anyattempts at detecting the organism. Another limitation of PCR is theinability of the investigator, without further study, to map and/orobtain sequence information about the amplified DNA (and consequentlythe isolated genomic material) other than information about thesequences complimentary to the two primers used in the analysis.Additionally, an automated inline PCR platform generally does notprovide a means to further analyze (e.g., map) genomic material after ithas been subjected to PCR. Further analysis (e.g., providing morecertain identification of a species and/or strain) of the genomicmaterial may be important and useful in, e.g., distinguishing apathogenic strain from a nonpathogenic strain, detecting and providingthe sequence of a new strain, choosing an appropriate antibioticregimen, etc.

To overcome some of the limitations of PCR, methods of waveformprofiling were developed (see, e.g., U.S. patent application Ser. Nos.11/190,942 and 11/356,807, and Japanese Patent Application PublicationNos. 2003-334082 and 2003-180351). Waveform profiling methods provideways to analyze and profile genomic material, e.g., DNA isolated fromorganisms, such as bacteria, without requiring the investigator to knowthe identity of the organism prior to detection.

Waveform profiling generally utilizes melting temperature analysisaccomplished with the use of detectible (e.g., radioactive, fluorescent,chemiluminescent, etc.) agents (e.g., nucleotides, intercalators, etc.)that are incorporated into higher-order DNA structures generated bywaveform profiling. As the temperature of the sample is increased, thehigher-order structures dissociate and, e.g., lose fluorescenceintensity (e.g., intercalated fluorescent agents dissociate). Plottingthe rate of change of fluorescence intensity obtained by thedissociation of these higher-order structures as a function ofincreasing temperature produces a waveform unique to the genomic DNA ofthe organism and the utilized waveform primer, i.e., the dissociation ofhigher-order DNA structures at different melting temperatures (Tm) isobserved and recorded to produce a characteristic “waveform profile” foreach species (or strain) of organism, e.g., bacteria. Accordingly,waveform profiling can be used to distinguish between genomic DNAisolated from a first organism and genomic DNA isolated from a secondorganism using melting temperature analysis. However, waveform profilingdoes not provide, without further investigation, the map or sequence ofthe analyzed genomic material.

Since waveform profiling is a relatively new method, advances describedherein can be used to increase its throughput and/or automation.

As discussed above, new technologies that increase the level of PCRthroughput and automation have been developed. An example of one suchtechnology is the use of microfluidic systems, includingcontroller/detector interfaces for such microfluidic systems, asdescribed in, e.g., U.S. Pat. Nos. 6,033,546; 6,238,538; 6,267,858;6,500,323; and 6,670,153. These microfluidic systems, collectivelyreferred to herein as automated inline PCR platforms, are well known inthe art and are generally described herein.

Most automated inline PCR platforms utilize a disposable microfluidicchip that works with controller/detector interfaces for automated sampleaccession, microfluidic PCR reagent assembly, PCR thermal cycling, andoptical detection spectroscopy. A microfluidic chip generally comprisesa first plate with at least one micro-etched fluidic (microfluidic)inline reaction channel that can be bonded to a second plate, withinwhich can be metal traces and a fluid reservoir. When the two plates arebonded together, each microfluidic reaction channel of the first platecan connect with a fluid reservoir of the second plate so thatlocus-specific reagents can be delivered through the fluid reservoirs tothe microfluidic inline reaction channels.

Inline PCR begins when a capillary, or “sipper,” aspirates a sampledroplet (which may or may not be a DNA sample droplet, i.e., a sampledroplet comprising genomic material isolated from an organism) from,e.g., a microtiter plate, into at least one microfluidic inline reactionchannel. After aspirating a sample droplet into a microfluidic inlinereaction channel, the sipper can be moved to a buffer trough so thatbuffer is drawn into the microfluidic chip. Consequently,cross-contamination among sample droplets is minimized or eliminatedsince each sample droplet is separated from adjacent sample droplets bybuffer spacers. Each sample droplet then moves along a microfluidicinline reaction channel and into a PCR assembly area of the chip,wherein the sample droplet becomes a sample plug by being mixed withPCR-required reagents, e.g., a primer pair, DNA polymerase, and dNTPs,and detectable agents, e.g., intercalators, etc. Optionally, bufferspacers can also be mixed with PCR-required reagents to serve asnegative controls. After being mixed with PCR-required and detectableagents, a sample plug (which may or may not be a DNA sample plug, i.e.,a sample plug comprising genomic material) moves along the length of themicrofluidic inline reaction channel into different areas of the chip,e.g., an amplification area wherein PCR can be effected on the sampleplugs.

Generally, as each sample plug (e.g., a DNA sample plug) flows through amicrofluidic inline reaction channel, it enters a temperature-controlledamplification area wherein each microfluidic inline reaction channel isrepeatedly and rapidly heated and cooled in a localized manner such thatthe denaturing, annealing and elongation steps of PCR are effected onthe sample plugs as they move through the channel(s); sample plugs thatdo not comprise genomic material are exposed to the same heating andcooling processes, etc. Amplification of DNA will occur only in DNAsample plugs, i.e., sample plugs comprising genomic material. A methodof controlling the temperature in the amplification area is Jouleheating (see, e.g., U.S. Pat. Nos. 5,965,410 and 6,670,153). Generally,voltage can be applied to the metal traces in or near the microfluidicinline reaction channel in a controlled and localized manner toeffectuate the different temperatures required for each PCR cycle.Cooling of the reaction can be achieved through the use of, e.g.,cooling fluid that travels through a coil to carry away thermal energyfrom the microfluidic inline reaction channel, rapid heat dissipation,e.g., by application of cold water to the bottom surface of themicrofluidic chip, or simple radiant convection into the atmosphere orsuitable heat transfer using a heat sink. Since the volume of fluid inthe microfluidic channels is small and the metal traces are located veryclose to the microfluidic inline reaction channels, heating and coolingof the fluid in the channels (and hence, sample plugs) is accomplishedvery rapidly. Consequently, DNA sample plugs undergo PCR, and PCR cyclesrun such that, e.g., 30 cycles can typically be performed in, e.g., lessthan nine minutes. The number of PCR cycles each DNA sample plug sees asit travels through a microfluidic channel in the temperature-controlledarea of the chip can be varied by changing, e.g., either or both 1) thetiming of the voltage applied to the metal traces, and 2) the flow rateof the DNA sample plugs through the microfluidic channels.

A microfluidic chip can simultaneously perform as many polymerase chainreactions as it has microfluidic inline reaction channels. For example,a sample comprising genomic material can be aspirated into multipledifferent microfluidic inline reaction channels, to each of which isadded a different locus-specific reagent (e.g., a different primer pairthat brackets a different locus on the genomic material, e.g., DNA).This permits simultaneously detecting several different loci of genomicmaterial isolated from the same organism. Alternatively, reagentscomprising one specific primer pair can be aspirated into multipledifferent microfluidic inline reaction channels. This permitssimultaneously detecting the same locus on genomic material isolatedfrom different samples and/or different organisms. Additionally,multiple sample droplets can be aspirated into the same microfluidicreaction channel.

A detection area is usually downstream of the temperature-controlledamplification area, and is generally a transparent region thatfacilitates observation and detection of the amplified DNA products,e.g., PCR products. In the detection area, each microfluidic inlinereaction channel is usually brought in close proximity and passed undera detector. A light source spreads light across the microfluidic inlinereaction channels so that detectable agents or energy, e.g.,fluorescence emitted from each channel, e.g., from each DNA sample plug,passing through the optical detection area can be measuredsimultaneously. After detection, each microfluidic inline reactionchannel usually directs each sample plug to a waste well.

Three different methods are usually used to generate fluid motion withinmicrofluidic inline reaction channels; the methods involveelectrokinetics, pressure, or a hybrid of the two (see, e.g., U.S. Pat.Nos. 6,238,538; 6,670,153; 6,787,088; and U.S. Published PatentApplication No. 2001/0052460) and nonmechanical valves (see, e.g., U.S.Pat. Nos. 6,681,788 and 6,779,559). In a pressure-based flow system, aninternal or external source can be used to drive the flow of fluid inthe inline reaction channels. For example, a vacuum can be applied towaste wells at the ends of each microfluidic inline reaction channel andcan be used to activate the sipper and move the fluid along themicrofluidic inline reaction channels toward the waste wells.Alternatively, since genomic material is charged, electrokinetics, i.e.,the generation of a voltage gradient (e.g., by the application ofvoltage to the metal traces) can be used to drive charged fluid alongthe microfluidic inline reaction channels. A third method of driving thefluid along the inline reaction channels uses both electrokinetics andpressure. The result is a continuous flow of fluid within themicrofluidic inline reaction channels, wherein sample plugs (e.g., DNAsample plugs) are continuously being mixed or moved to different areas(e.g., a PCR assembly area, a temperature-controlled area, a detectionarea, etc.) of the chip.

Electrokinetic and/or pressure-driven fluid movement, heating andcooling cycles, detection, and the data acquisition related to amicrofluidic chip can be controlled by an instrument that interfaceswith the chip (generally described in, e.g., U.S. Pat. Nos. 6,033,546and 6,582,576). The interface of the instrument usually contains o-ringseals that seal the reagent wells on the chip, pogo pins that caninterface with the metal traces on the chip and supply the voltage fortemperature cycling, o-ring seals for the waste wells where a vacuum canbe applied to move the fluid through the chip, a large o-ring that canbe used to seal the bottom of the chip against circulating cool waterand to speed the cooling during the temperature cycling, and a detectionzone for, e.g., fluorescence detection. The risk of contamination withthis system is minimal because a microfluidic chip is usually a closedsystem with physical barriers (e.g., buffer spacers) separating DNAsample plugs. Moreover, continuous flow prevents sample plugs frommoving backwards.

The automated inline PCR platforms described above are limited in thatthe microfluidic chips should be disposed of after use and are notsuitable for automated inline waveform profiling; also, analyzingsamples using such a platform requires outsourcing. Additionally, theuse of a sipper to aspirate sample droplets is an inefficient andwasteful method to obtain the small volume required to effect PCR cyclesrapidly. The present invention resolves these limitations by providing amolecular diagnostic device that can be used to characterize genomicmaterial isolated from an organism (e.g., bacteria, viruses) in a sampleby automated methods of preparing the genomic material, and then eitheror both 1) amplifying the genomic material and detecting any amplifiedproducts and 2) mapping the genomic material. A molecular diagnosticdevice of at least one exemplary embodiment of the invention has theadvantage that multiple samples, e.g., patient samples, can be processedthrough the same microfluidic chip without cross-contamination. Also,because in some exemplary embodiments the device is a portable system, adevice of at least one exemplary embodiment of the invention can beutilized at different patient care centers throughout the Unites Statesor elsewhere in the world, and can also be used in a near-patientsetting or a contaminated site away from a hospital or other patientcare center. The molecular diagnostic device disclosed herein also hasthe advantage of facilitating the screening of samples within a shorttime after collection.

SUMMARY OF THE INVENTION

At least one exemplary embodiment is directed to a cartridge configuredfor isolating genomic material, wherein the cartridge comprises at leastone channel and a solid substrate capable of binding and releasinggenomic material. In at least one further exemplary embodiment, thecartridge further comprises a waste well. In another exemplaryembodiment, the solid substrate capable of binding and releasing genomicmaterial (i.e., binding and release substrate) comprises charge switchmaterial.

At least one exemplary embodiment of the invention is directed to acartridge configured to isolate genomic material comprising: a reactionchamber, and at least one binding and release substrate, wherein the atleast one binding and release substrate lies within the reaction chamberand is configured to bind and release at least a portion of a samplecomprising genomic material. In at least one other exemplary embodiment,the substrate comprises charge switch material. In at least one furtherexemplary embodiment, the binding and releasing of at least a portion ofthe sample is in response to an electric voltage. In at least one otherfurther exemplary embodiment, the binding and releasing of at least aportion of the sample is in response to a difference in ioniccomposition of a fluid in contact with the substrate, and the sample iscontained within the fluid. In at least one other further exemplaryembodiment, the binding and releasing of at least a portion of thesample is in response to a difference in pH of a fluid in contact withthe substrate, and the sample is contained within the fluid. In at leastone other exemplary embodiment, the substrate is a particle or bead. Inat least one other further exemplary embodiment, the substrate ismagnetic or paramagnetic. In at least one other further exemplaryembodiment, the substrate is bound to the inner surface of the reactionchamber. In at least one other exemplary embodiment, the volume of atleast a portion of the sample released from the substrate is reducedfrom the volume of the sample.

At least one exemplary embodiment of the invention is directed to acartridge, configured to deliver a sample comprising genomic material,comprising a genomic separation and direction system, and an ejectorhead, wherein a chamber in the ejector head is configured to receive atleast a portion of a sample comprising genomic material from the genomicseparation and direction system, and wherein the ejector head expels atleast a portion of the received sample out of the cartridge as ejectedsample droplets. In at least one other exemplary embodiment, the ejectorhead uses thermal energy provided by a thermal energy generator. In atleast one further exemplary embodiment, the ejector head is a piezo jetsystem.

At least one exemplary embodiment of the invention is directed to acartridge configured to isolate genomic material comprising a reactionchamber, at least one binding and release substrate, wherein the atleast one binding and release substrate lies within the reaction chamberand is configured to bind and release at least a portion of a samplecomprising genomic material in response to an electric voltage, agenomic separation and direction system, and an ejector head, wherein achamber in the ejector head is configured to receive at least a portionof the sample from the genomic separation and direction system, andwherein the ejector head expels at least a portion of the receivedsample out of the cartridge as ejected sample droplets. In at least oneother exemplary embodiment, the substrate comprises charge switchmaterial. In at least one further exemplary embodiment, the substrate isa particle or bead. In at least one other further exemplary embodiment,the substrate is magnetic or paramagnetic. In at least one otherexemplary embodiment, the substrate is bound to the inner surface of thereaction chamber. In at least one other exemplary embodiment, theejector head uses thermal energy provided by a thermal energy generator.In at least one further exemplary embodiment, the ejector head is apiezo jet system.

At least one exemplary embodiment of the invention is directed to amolecular diagnostic device comprising at least one cartridge and atleast one microfluidic chip, wherein the chip is configured to receiveat least a portion of the sample droplets ejected from the cartridge,and wherein the chip comprises at least one microfluidic inline reactionchannel for receiving the sample droplets ejected from the cartridge. Inat least one other exemplary embodiment, the ejector head of thecartridge can be repetitively pulsed to form multiple droplets insequence at a repetitive pulse rate to achieve a controlled totaldroplet volume in the microfluidic inline reaction channel of themicrofluidic chip. In at least one further exemplary embodiment, therepetitive pulse rate is in the range of about 1 kHz to about 100 kHz.In at least one other further exemplary embodiment, the repetitive pulserate is about 50 kHz. In at least one other exemplary embodiment, anejected sample droplet has a volume in the range of about 1 picoliter toabout 25 picoliters. In at least one other further exemplary embodiment,the ejected sample droplet has a volume of about 3 picoliters. In atleast one other exemplary embodiment, the total droplet volume is in therange about 3 picoliters to about 100 nanoliters. In at least onefurther exemplary embodiment, the microfluidic chip further comprises anamplification area within a first temperature-controlled area for theamplification of DNA products, and a detection area within a secondtemperature-controlled area, and detection of amplified DNA products canoccur at more than one temperature. In at least one other exemplaryembodiment, the device further comprises a matrix analysis area.

At least one exemplary embodiment of the invention is directed to amicrofluidic chip comprising a sample droplet receiving systemconfigured to receive at least a portion of sample droplets comprisinggenomic material ejected by a cartridge, and a matrix analysis area,wherein the matrix analysis area comprises an emitter layer, a filterlayer, and a detector layer, wherein the emitter layer emits an emitterwavelength. In at least one other exemplary embodiment, the filter layercomprises an optical filter doped glass that passes a fluorescentwavelength and blocks the emitter wavelength. In at least one otherexemplary embodiment, the microfluidic chip further comprises at leasttwo channels, wherein the two channels are configured to flow samplescomprising genomic material through an amplification area within a firsttemperature-controlled area for the amplification of DNA products, and adetection area within a second temperature-controlled area for theinitiation of fluorescence of DNA products, and wherein detection ofamplified DNA products can occur at more than one temperature.

At least one exemplary embodiment of the invention is directed to amolecular diagnostic device comprising at least one cartridge and amicrofluidic chip, wherein the cartridge ejects sample dropletscomprising genomic material into the sample droplet receiving system ofthe microfluidic chip. In at least one other exemplary embodiment, thematrix analysis area further comprises more than one unit, and each unitcomprises at least one photon generator component, a DNA stretchchip,and at least one photon detector component. In at least one furtherexemplary embodiment, the at least one photon detector componentcomprises porphyrin gate material. In at least one other exemplaryembodiment, the at least one photon detector component comprises threethin-film transistors. In at least one other further exemplaryembodiment, the device is portable. In at least one further exemplaryembodiment, the device is hand-held.

At least one exemplary embodiment of the invention is directed to amethod of characterizing genomic material in a sample, comprising thesteps of (a) isolating any genomic material in the sample with acartridge; (b) ejecting at least one sample droplet from a liquidejection mechanism in the cartridge into a sample droplet receivingsystem of a microfluidic chip; (c) detecting genomic material in thesample droplet; and (d) analyzing the sample droplet to characterize thegenomic material present. In at least one other exemplary embodiment,analyzing the sample droplet comprises comparing a detected barcode fromthe genomic material in the sample with a database of known barcodes.

At least one exemplary embodiment of the invention is directed to amolecular diagnostic device comprising at least one cartridge forisolating genomic material; at least one sample droplet ejection headfor ejecting the genomic material from the cartridge after it isisolated, wherein the at least one cartridge can be attached to at leastone sample droplet ejection head; and at least one microfluidic chip foranalyzing the genomic material, wherein the microfluidic chip comprisesat least one microfluidic inline reaction channel for receiving theejected genomic material from the sample droplet ejection head and atleast one metal trace for heating of and/or fluid movement within themicrofluidic inline reaction channel, and wherein the at least onemicrofluidic inline reaction channel runs through a reagent assemblyarea, an amplification area within a first temperature-controlled areafor the amplification of DNA products, and a detection area. In at leastone further exemplary embodiment, the detection area is within a secondtemperature-controlled area, and the detection of amplified DNA productscan occur at more than one temperature. In, another exemplaryembodiment, the device further comprises a matrix analysis area. In afurther exemplary embodiment, the matrix analysis area comprises morethan one unit, and each unit comprises at least one photon generatorcomponent, a DNA stretchchip, and at least one photon detectorcomponent. In another exemplary embodiment of the invention, themolecular diagnostic device is portable. In another further exemplaryembodiment, the matrix analysis area of the molecular diagnostic devicecomprises a matrix of 512×512 units.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a cartridge design in accordance with at least oneexemplary embodiment of the invention, and FIG. 1B illustrates acartridge interfacing with a chip in accordance with at least oneexemplary embodiment.

FIG. 2 schematically diagrams ejection heads and microfluidic ports infixed positions relative to each other in accordance with at least oneexemplary embodiment.

FIG. 3 schematically diagrams a massive sample partitioning of amicrofluidic inline reaction channel into multiple subchannels inaccordance with at least one exemplary embodiment.

FIG. 4 schematically delineates a sample droplet path as it is mixedwith amplification reagents to form a sample plug in the reagentassembly area and is amplified within the amplification area inaccordance with at least one exemplary embodiment.

FIG. 5 schematically delineates a sample plug path in a microfluidicinline reaction channel after it has passed the temperature-controlledarea of FIG. 4 and passes through a detection area in accordance with atleast one exemplary embodiment.

FIGS. 6A-6C depict several aspects of a timing chart of at least oneexemplary embodiment of the invention. FIG. 6A depicts a representationof a 2×2 matrix, including representations of row wiring (RW) and columnwiring (CW), as well as the timing chart (Timing Chart 2) for the clockpulses OUT1 and OUT2 from a binary shift register (BSR1). FIG. 6Bdepicts Timing Charts 1 and 4, and FIG. 6C depicts Timing Chart 3 (whichfollows the status of pulses IN1 and IN2 from BSR2 (as shown in FIG.6A)).

FIG. 7 is a cross-section of a single channel microcapillary (i.e.,microfluidic) component of the matrix analysis area of the device inaccordance with at least one exemplary embodiment.

FIG. 8 is an optical diagram generally showing the principle ofoperation in use as a fluorescence detection system in accordance withat least one exemplary embodiment.

FIG. 9 illustrates the implementation of the matrix (or array) withinthe matrix analysis area in accordance with at least one exemplaryembodiment.

FIG. 10 is a top view of a 90-channel microfluidic array with matrixedoptical detection zones in accordance with at least one exemplaryembodiment.

FIG. 11 shows glass filter wavelength characteristics.

DETAILED DESCRIPTION OF THE INVENTION

The following description of at least one exemplary embodiment is merelyillustrative in nature and is in no way intended to limit the invention,its application, or uses.

Processes, techniques, apparatus, and materials as known by one ofordinary skill in the relevant art may not be discussed in detail butare intended to be part of the enabling description where appropriate,for example, the fabrication of microfluidic channels, subchannels, andmicrochannels and their related materials.

In all of the examples illustrated and discussed herein, any specificvalues, for example, the amount of fluid (e.g., picoliters), should beinterpreted to be illustrative only and nonlimiting. Thus, otherexamples of the exemplary embodiments could have different values.

Note that similar reference numbers and letters refer to similar itemsin the figures disclosed and discussed herein, and thus once an item isdefined in one figure, it may not be discussed for following figures.

At least one exemplary embodiment of the present invention is directedto a molecular diagnostic device and methods of using the moleculardiagnostic device for automated methods of characterizing genomicmaterial in a sample. In a broad exemplary embodiment, the methodentails preparing sample genomic material (i.e., isolating any material,and portioning the sample(s)) and identifying the genomic material by(either or both) 1) amplification and detection of amplified productsand 2) mapping. The molecular diagnostic device of at least oneexemplary embodiment of the invention comprises at least one cartridgeand at least one liquid ejection mechanism for the preparation(isolation and partitioning of genomic material of a sample to beanalyzed); and at least one microfluidic chip for amplification ofgenomic material isolated from the sample and detection of productsamplified from the genomic material. In at least one exemplaryembodiment of the invention, the liquid ejection mechanism transfers aprepared sample from the cartridge to the microfluidic chip. In anotherexemplary embodiment of the invention, a molecular diagnostic device ofthe invention further comprises a matrix analysis area for mapping ofthe genomic material. Thus, a molecular diagnostic device of at leastone exemplary embodiment of the invention can be used for an automatedmethod of characterizing genomic material, the method comprising thesteps of preparing the genomic material and either or both 1) amplifyingthe genomic material and detecting the amplified products and 2) mappingthe genomic material.

It will be easily recognized that for purposes herein, the step ofpreparing a sample comprises isolating genomic material, if present,from the sample, and partitioning the sample (including any isolatedgenomic material) for analysis. Also the step of amplifying the genomicmaterial comprises mixing any isolated genomic material with appropriateamplification reagents (e.g., primers, dNTPs, salts, buffers, etc.) andoptionally detection reagents (e.g., intercalators) and effecting anamplification reaction on the isolated genomic material. Nonlimitingexamples of amplification reactions that can be performed with amolecular diagnostic device of at least one exemplary embodiment of theinvention include PCR and various forms of waveform profiling (see,e.g., U.S. patent application Ser. Nos. 11/190,942 and 11/356,807, thecontents of which are hereby incorporated by reference herein in theirentireties). The step of detecting is dependent on the amplificationmethod used, i.e., whether a PCR product or the dissociation ofhigher-order structures is detected depends on whether PCR or a waveformprofiling method, respectively, is performed. After a sample isprepared, a molecular diagnostic device of at least one exemplaryembodiment of the invention will permit any isolated genomic materialto 1) be amplified and detected via its amplified products, 2) beamplified and detected via its amplified products, and mapped within thematrix analysis area, described herein, or 3) be mapped within thematrix analysis area, described herein.

Molecular Diagnostic Device

Over the last few years, automated inline PCR platforms have beendeveloped for compatibility with a variety of existing and well-knownfluorescent “mix-and-read” biochemistries, such as TaqMan, MolecularBeacons, Epoch Eclipse Probes, and Allele Specific Amplification. Todate, no known automated inline platform has allowed efficient on-site(e.g., near-patient) genetic testing that would obviate the need tooutsource samples, e.g., patient samples collected at a doctor's officeor at another near-patient site. It is an object of the invention toprovide a molecular diagnostic device that can be used, e.g., to analyzea patient sample for, e.g., the presence of bacterial or viralinfection, at the same place and within a short time frame (e.g.,approximately one hour) of the procurement of the sample to be analyzed.As described in further detail herein, a molecular diagnostic device ofat least one exemplary embodiment of the invention comprises at leastone cartridge configured for isolating any genomic material present in asample; at least one liquid ejection mechanism comprising at least oneejection head for partitioning any isolated genomic material, whereinthe at least one cartridge can be or is temporarily attached to the atleast one ejection head; and at least one microfluidic chip fordetecting any genomic material, wherein the microfluidic chip comprisesat least one microfluidic inline reaction channel for receiving theejected genomic material from the liquid ejection mechanism and at leastone metal trace for heating of and/or fluid movement within themicrofluidic inline reaction channel, and wherein the at least onemicrofluidic inline reaction channel runs through a reagent assemblyarea, an amplification area within a first temperature-controlled areafor the amplification of DNA products, and a detection area of themicrofluidic chip. In another exemplary embodiment of the invention, amolecular diagnostic device of the invention further comprises a matrixanalysis area for genomic mapping.

1. Preparing Genomic Material

The molecular diagnostic device of at least one exemplary embodiment ofthe invention comprises a cartridge and a liquid ejection mechanism,both of which are involved particularly in the step of preparing anygenomic material present in a sample. In other words, the genomicmaterial can be isolated from a collected sample (e.g., a patientsample) via a cartridge and subsequently ejected via the liquid ejectionmechanism prior to the later steps of amplifying and detecting (and/ormapping) via a microfluidic chip.

It is contemplated by the inventors that a molecular diagnostic deviceof at least one exemplary embodiment of the invention can be used foron-site (e.g., near-patient) testing of patient samples; in addition,many different types of samples can be tested using a moleculardiagnostic device of the invention. Such samples include, but are notlimited to, water, air, dust, food, and biological samples, includingbody fluids (e.g., saliva, whole blood, plasma, buffy coat, urine,etc.), cells (e.g., whole cells, cell fractions, and cell extracts), andtissues. Biological samples also include sections of tissue such asbiopsies and frozen sections taken, e.g., for histological purposes.Exemplary biological samples include, but are not limited to, blood,plasma, lymph, tissue biopsies, urine, CSF (cerebrospinal fluid),synovial fluid, and BAL (bronchoalveolar lavage). In at least oneexemplary embodiment of the invention, the biological sample is blood.

A. Cartridge

The sample can be collected utilizing any well-known method(s), e.g., asyringe for the collection of a patient blood sample, and then placedinto a disposable cartridge of at least one exemplary embodiment of theinvention for the isolation of genomic material from the sample; in atleast one exemplary embodiment, the sample is collected directly intothe disposable cartridge. A cartridge of at least one exemplaryembodiment of the invention is configured to isolate any genomicmaterial contained in a sample and comprises at least one channel(and/or chamber) within the cartridge; genomic material is isolated froma sample in the at least one channel within the cartridge.

FIG. 1A illustrates a nonlimiting example of a cartridge 100 a inaccordance with at least one exemplary embodiment, and FIG. 1Billustrates the interaction between a cartridge 100 b and a chip 190.The cartridge 100 a illustrated in FIG. 1A includes: an external sampleinlet 110; optionally an inlet cap 115; a sample chamber A; a push valveB1 that can be pushed to allow an external sample to enter the samplechamber A (e.g., which can be evacuated via a pump inlet F1, thensealed, to have a lower pressure to initiate an external sample to enterchamber A); optional electrodes 140 (e.g., to provide a voltagedifference to break up cell membranes or cell walls to release thegenomic material into or within chamber A); a push valve B2 that can bepushed to allow the genomic material from chamber A to enter a reactionchamber B; where binding and release substrates 150 can bind and releasegenomic material; where optional electrodes 160 can be used to directthe genomic material (e.g., genomic material extracted from the sample)into channel D, while waste material can be directed via channel C outthrough waste outlet 13; where a printhead 130 collects genomic materialin a printhead chamber E and can be used to eject material intochannels, e.g., microfluidic inline reaction channels, e.g., positionedflush with the cartridge (not shown); where wash can be inserted via awash insertion inlet I1; where reagent can be inserted via a reagentinsertion inlet 12; where a reagent insert valve can be pushed B3 toallow reagent to enter the reaction chamber B; where a wash insert valvecan be pushed B4 to allow wash to enter the reaction chamber B; andwhere an optional power system 120 can supply the power to the printhead130, and electrodes 140 and 160, and binding and release substrates 150.In at least one exemplary embodiment of the invention, the power system120 supplies power to a thermal energy generator connected to orcomprised within the printhead 130. In at least one exemplary embodimentof the invention, the path of released genomic material moving fromreaction chamber B through channel D leading to printhead chamber E,along with the associated movement of the genomic material andassociated electrodes 160 optionally controlling the movement of thegenomic material, is referred to as a genomic separation and directionsystem.

Note that FIG. 1A illustrates only one nonlimiting example; variationsare intended to lie within other exemplary embodiments. For example, thepower system 120 can be external, the reagents and wash can lie ininternal cambers in the cartridge, the push valves can be replaced withother types of methods to prohibit and allow fluid passage as known byone of ordinary skill in the relevant arts (including push valves andother devices or methods controlled by electronic means), the wastematerial can be stored in an internal chamber in the cartridge, and,although FIG. 1A illustrates genomic material from one sample beinginjected, multiple insertions from various samples from, e.g., differentindividuals (e.g., patients), or other sources (e.g., a water supply)can be inserted by, e.g., duplicating parts of the cartridge alreadydescribed, e.g., one for each person (e.g., patient) or other source(e.g., a water supply) sampled.

Although there is no limitation to the amount of sample that can becollected into a cartridge of the invention, it is contemplated that thecartridge be used to determine genomic material from approximately 100μl of sample. For example, volumes for preparation of a sample by acartridge of at least one exemplary embodiment of the invention can bein the range of 10 μl to 1 ml.

In a cartridge of at least one exemplary embodiment of the invention,genomic material is isolated from a sample using only a homogeneousaqueous solution. Use of a homogeneous solution conveniently obviatesthe inefficient requirement of most methods of isolating genomicmaterial (e.g., switching between alcohol-based or other organic-basedsolutions and aqueous solutions, and centrifugation), and alsofacilitates the use of the cartridge with the liquid ejection mechanismof exemplary embodiments of the present invention. Also unlike methodswhere genomic material is bound to a substrate, the cartridge of thepresent invention (incorporating the “charge switch” technologydescribed herein) allows the genomic material to be bound to a substrateunder one condition or released from the substrate under anothercondition. Thus, it is within the scope of the invention that acartridge can, in some exemplary embodiments, be reused to isolategenomic material from more than one sample.

For example, in at least one exemplary embodiment, a sample can becollected, diluted to achieve a certain volume (e.g., in an aqueouslysis buffer), and simultaneously or subsequently drawn into a cartridgeof the invention (e.g., into a reaction chamber of the cartridge), theconditions of which promote binding of the genomic material to thesurface of a binding and release substrate (also referred to herein assubstrate). In another exemplary embodiment, the aqueous lysis buffercan be diluted within the cartridge. After binding of the genomicmaterial to the binding and release substrate, unbound macromolecules(e.g., proteins, contaminants, etc) are washed away. The condition ofthe cartridge can then be changed to release (i.e., elute) the genomicmaterial into, e.g., an aqueous buffer solution or water. The eluatecomprising genomic material can then be sent via a liquid ejectionmechanism, as described herein, to a microfluidic chip for analysis. Inat least one exemplary embodiment of the invention, the cartridge isdisposed after it is used with a sample. In another exemplary embodimentof the invention, the cartridge can be washed, including subjecting thesurface of a substrate of the cartridge to conditions that promotebinding and/or release of genomic material, and reused to isolategenomic material from another sample.

For purposes of a cartridge of at least one exemplary embodiment of theinvention, a binding and release substrate can be any suitable supportwith a solid surface, e.g., particles, beads, tubes, wells, glass,plastic, etc. A suitable support with a solid surface for a cartridge ofat least one exemplary embodiment of the invention is such that it canhave a natural affinity for genomic material or it has been or is easilyconditioned (e.g., with conditioning buffer) for the binding and releaseof genomic material within the cartridge. Suitable methods forconditioning the surface of a solid-phase support include treating itwith conditioning buffer (e.g., a substance that can introduce a charge(e.g., a positive charge) on the surface, or cause the surface to behydrophilic or hydrophobic, etc.). Additionally, a suitable support canbe magnetizable, magnetic, paramagnetic, etc. In at least one exemplaryembodiment, the inner surface of the cartridge includes the innersurface of a channel(s) within the cartridge. In at least one furtherexemplary embodiment of the invention, the inner surface of thecartridge can be used as the substrate. In another exemplary embodiment,particles or beads (including magnetized or magnetizable particles orbeads) within the cartridge can be used as the substrate.

For example, U.S. Published Patent Application Nos. 2003/0054395 and2003/0130499, each of which are incorporated herein by reference intheir entireties, describe methods of isolating genomic material in anaqueous solution comprising conditioning a solid phase substrate to bindand subsequently release genomic material. Briefly, these applicationsdescribe providing the surface of a solid phase substrate (e.g., anonporous solid-phase substrate) with a charge that can be switcheddepending on the conditioning buffer, i.e., “charge switch material,”that is in, on, or actually comprises the solid phase substrate.According to U.S. Published Patent Application No. 2003/0054395, chargeswitch materials are ionizable. For example, chemical species comprisingionizable groups can be immobilized onto solid supports in monomeric orpolymeric form via adsorption, ionic or covalent bonds, or covalentattachment to a polymer backbone, which, in turn, is immobilized onto asolid support. Alternatively, chemical species can be incorporated intosolid and insoluble forms, e.g., beads, particle, paths, channels, etc.(e.g., within a cartridge). The charge switch material is generallychosen so that the pKa of the ionizable group is appropriate to theconditions at which it is desired to bind and release nucleic acids fromthe solid phase substrate. Generally, genomic material will bind to thecharge switch material at a pH below or roughly equal to the pKa, whenthe charge switch material is positively charged, and will be releasedat a higher pH (usually above the pKa), when the charge switch materialis less positively charged, neutral, or negatively charged. In otherwords, in conditions of low pH, the substrate surface has a positivecharge that is able to bind to negatively charged genomic material.Contaminants, e.g., proteins, other macromolecules, etc., are not boundand can be washed away in an aqueous wash buffer at normal physiologicaltemperatures. Increasing the pH will neutralize the charge of thesubstrate surface and effect the release of genomic material, which canbe eluted with an aqueous elution buffer.

Consequently, in at least one exemplary embodiment of the invention, theinner walls of the cartridge, or of a channel(s) within the cartridge,can be a solid phase substrate provided with a charge that can beswitched depending on the condition of pH. In another exemplaryembodiment, small solid phase particles or beads (e.g., magnetic beads)to which a switchable charge can be provided are within the cartridge.This latter exemplary embodiment provides an appropriate mechanism forpreventing the beads from being washed away with the contaminants, e.g.,by application of a magnetic field. In at least one exemplaryembodiment, an optional power system within or external to thecartridge, supplies power to effect changes in the state of the bindingand release substrate(s) of the invention.

Fluid (e.g., conditioning buffer, sample, lysis buffer, contaminants,eluate, wash buffer, etc.) can take different paths within the cartridgefor the isolation of genomic material, and ultimately, the ejection ofsample droplets that may contain isolated genomic material. For example,the sample can proceed along a straight, winding, or tubular path (whichcan be of any three-dimensional shape (e.g., a tube that is circular,semi-circular, square, etc. in cross-section)), join another path (e.g.,to be mixed with lysis buffer), separate into two or more other pathswithin the cartridge, be allowed to pool, mix, and/or incubate withother materials, etc. In at least one exemplary embodiment of theinvention, capillary action is used to draw the sample, conditioningbuffer, lysis buffer, contaminants, eluate, wash buffer, etc., throughthe cartridge to a liquid ejection mechanism of at least one exemplaryembodiment of the invention. Capillary action may require that thecartridge be filled with fluid (e.g., with conditioning buffer) prior tothe introduction of the sample; in another exemplary embodiment,pressure from the injection of the sample (e.g., blood) can alone besufficient to propel the sample through the cartridge. In a furtherexemplary embodiment of the invention, a vacuum is applied to draw thesample through the cartridge. In still another exemplary embodiment,electrokinetic forces move the sample, or portions of the sample (e.g.,genomic material extracted from the sample), through the cartridge.

Another component of the cartridge is its ability to reduce the volumeof the patient sample. In at least one exemplary embodiment of theinvention, any isolated genomic material is eluted in a volume of liquidthat is less than the original volume of the sample. In at least onefurther exemplary embodiment of the invention, any isolated genomicmaterial is eluted in microliter, e.g., about 1-10 μl, e.g., about 2.5μl, volumes.

As the methods herein describe DNA amplification processes, if thegenomic material isolated is RNA, it must first be reverse transcribedinto DNA, e.g., cDNA, prior to amplification via the microfluidic chip.Methods of reverse transcription are well known in the art. In at leastone exemplary embodiment, the genomic material isolated is the entiregenomic DNA of an organism. In another exemplary embodiment, genomic DNAis purified to the exclusion of RNA using well-known reagents, e.g.,RNase.

A skilled artisan will recognize the desirability for isolationtechnology, e.g., wherein sample droplets are maintained separately fromeach other (e.g., a sample droplet is isolated and distinct from thesample droplet before it and the sample droplet after it), particularlyfor molecular diagnostics. Although most automated inline platformsutilize a sipper for such isolation, i.e., to draw a prepared sampleinto a microfluidic chip, use of a sipper is wasteful since the entiresample in the well is not examined. Additionally, sippers do not lendthemselves well to massive sample partitioning, i.e., partitioning asample with a small volume, e.g., about 2.5 μl, into, e.g., about onethousand isolated (sample) droplets. The present invention resolves thelimitations of a sipper and facilitates massive sample partitioning viaa liquid ejection mechanism. For example, a cartridge of at least oneexemplary embodiment of the invention can be attached or connected in anairtight manner to a liquid ejection mechanism for ejecting sampledroplets, including DNA sample droplets, by way of a liquid jet systemthat is adapted to eject sample droplets into, or across an airspace tobe received by, a microfluidic inline reaction channel of a microfluidicchip.

B. Liquid Ejection Mechanism

As noted, a cartridge of at least one exemplary embodiment of theinvention comprises, or is provided with a connection(s) to, a liquidejection mechanism for ejecting sample droplets, including DNA sampledroplets, by way of a liquid jet system. The liquid ejection mechanismin turn can comprise one or more liquid ejecting sections, i.e., anejection head (also referred to herein as a printhead). The number ofejection heads is typically predetermined by the number of samples to betested or the number of microfluidic inline reaction channels in amicrofluidic chip. Each of the liquid ejecting (or liquid ejection)heads is provided with a liquid-containing reservoir section forcontaining a prepared sample, an ejection port fluidly communicatingwith the liquid reservoir section (if necessary, together with a liquidpath for communicating the liquid reservoir section with thecorresponding ejection port), and an energy-generating mechanismprovided adjacent to the ejection port. This arrangement makes possiblemassive partitioning of the prepared sample by ejecting sample dropletsindependently of the prepared sample remaining in the liquid-containingsection.

An ejection head of at least one exemplary embodiment of the inventionis adapted to eject or expel sample droplets, typically utilizingthermal energy provided by a thermal energy generator. A conventionalliquid jet system can be used, such as a bubble jet system that ejectsliquid by generating bubbles (e.g., fluid bubbles) using thermal energyfrom electrothermal converters, such as heaters or lasers. In addition,in at least one exemplary embodiment of the invention, the ejector headis a piezo jet system. Thus, a conventional piezo jet system that ejectsliquid by applying a voltage to the piezo-element can be utilized. Theejection head to be used with the thermal jet system has a relativelysimple structure as compared with the head in the piezo jet system, andhence, can more easily be downsized and provided with a multi-nozzle.Additionally, the time required for forming the sample droplets fordelivery to the microfluidic inline reaction channel (or port) isrelatively short, so that production of a secondary structure of DNA bythe heat can be avoided, and the efficiency of subsequent hybridization,e.g., to amplification primers, can be improved. Thus, the thermal jetsystem is advantageously used as the liquid jet system for the purposeof the present invention.

Typically, it is advantageous for the purpose of the invention to applythe basic principles in the field of liquid jet recording for projectingdroplets of liquids (e.g., as disclosed in U.S. Pat. Nos. 4,723,129 and4,740,796) to sample droplet ejection. Both disclosed principles ofso-called on-demand type and continuous-type processes can be appliedherein. However, it may be advantageous to use the on-demand typeprocess for the purpose of this invention to minimize thermal energygenerated in the electro-thermal converters arranged in correspondenceto the liquid paths. Consequently, fluid bubbles can be formed in theprepared sample in one-to-one correspondence relative to the drivesignal. A prepared sample(s) is ejected by way of an ejection port as aresult of growth and contraction of the bubble to produce at least onesample droplet. If the drive signal is repetitive (e.g., 1 kHz to 100kHz′ e.g., 50 kHz), multiple picoliter drops can be dispensed in a shorttime frame to generate a fluid bubble of any practical size and volumeneeded for the particular analysis. Since the volume ratio of the fluidbubble to the encompassing medium can be controlled, known diluteconcentrations can be achieved when dispensing sample droplets (e.g.,sample droplets comprising genomic material and reagents). Apulse-shaped drive signal, as described in U.S. Pat. Nos. 4,463,359 and4,345,262 can suitably be used for these purposes. In at least oneexemplary embodiment of the molecular diagnostic device of theinvention, the ejector head of the cartridge can be repetitively pulsedto form multiple droplets in sequence at a repetitive pulse rate toachieve a controlled total droplet volume in the microfluidic inlinereaction channel of the microfluidic chip (e.g., in the microfluidicport of the microfluidic inline reaction channel). In at least one otherexemplary embodiment of the invention, the repetitive pulse rate is inthe range of about 1 kHz to about 100 kHz. In at least one furtherexemplary embodiment, the repetitive pulse rate is about 50 kHz.Ejections of sample droplets can be further improved when conductedunder well-known conditions, e.g., as described in U.S. Pat. No.4,313,124.

In at least one exemplary embodiment of the invention, an ejected sampledroplet has a volume in the range of about 1 picoliter to about 25picoliters. In at least one other exemplary embodiment, the volume ofthe ejected sample droplet is 3 picoliters. In at least one furtherexemplary embodiment of the invention, the total sample droplet volume(e.g., the summation of ejected sample droplets as received together in,e.g., the microfluidic port of a microfluidic inline reaction channel)is in the range of about 3 picoliters to about 100 nanoliters. In atleast one additional exemplary embodiment of the invention, the totalsample droplet volume is in the range of about 20 picoliters to about 10nanoliters.

As for the configuration of the ejection head, those configurationstaught by U.S. Pat. Nos. 4,558,333 and 4,459,600 are within the scope ofthe present invention. Additionally, the advantages of the presentinvention can be more effectively attained by providing a common slitfor the ejection sections of a plurality of electro-thermal converters(as disclosed in Japanese Patent Application Laid-open No. 59-123670)and arranging an open hole for absorbing pressure waves (as disclosed inJapanese Patent Application Laid-open No. 59-138461). In short,regardless of the particular configuration of the liquid ejection head,capture of sample droplets by a microfluidic inline reaction channel canbe accurately and efficiently realized according to the presentinvention.

Furthermore, a serial-type liquid ejection head rigidly secured to theapparatus main body, a replaceable tip-type liquid ejection head that iselectrically connected to the apparatus main body, or a cartridge-typeliquid ejection head provided with an integral solution reservoir can beadvantageously utilized.

In at least one exemplary embodiment of the invention, a liquid ejectionmechanism comprising a liquid ejection head that is provided with anejection-restoring apparatus and/or a spare auxiliary apparatus is used.Specific examples include a cleaner to be used for the liquid ejectionhead, a pressurizing or suction device, a spare heater that can be anelectro-thermal converter or a heating element of a different type or acombination thereof, and a spare ejector that is adapted to eject liquidin a form other than spotting.

Liquid jet systems generally generate waste in the form of satellitedroplets that are smaller than the sample droplets. In at least oneexemplary embodiment, these satellite droplets are angled away from anymicrofluidic inline reaction channel to prevent contamination. Theairspace between any ejection head and any microfluidic inline reactionchannel can be utilized to prevent contamination by satellite dropletsby providing a vacuum or another suitable device to draw satellitedroplets away from a microfluidic inline reaction channel and toward,e.g., any well-known and suitable waste capture mechanism. In addition,the waste capture mechanism can be used to capture waste from thecartridge, e.g., conditioning buffer, waste fluids from cleaning, etc.

In at least one exemplary embodiment, the design of the liquid ejectionmechanism allows an ejection head and a microfluidic inline reactionchannel to be aligned in a manner that facilitates ejection of sampledroplets from the liquid ejection head across an airspace into amicrofluidic inline reaction channel. For example, in at least onefurther exemplary embodiment, both the at least one microfluidic inlinereaction channel and the at least one ejection head are in a fixedposition relative to each other. However, the ejection head can stillrotate to point in different directions, e.g., to point toward a wastecapture device. Also, the microfluidic inline reaction channel can bedesigned to accept sample droplets from various angles (e.g., the entryto the microfluidic inline reaction channel can have a funnel-likeopening) with plural ejection heads aimed at the channel entry. In suchan exemplary embodiment(s), one or more sample droplet ejection headscan be placed at different angles in relation to the direction of thedroplet movement from respectively attached cartridges (e.g., thevarious different angles allow more than one ejection head to supplysample droplets to a single microfluidic inline reaction channel). It iswithin the scope of the invention that at least one cartridge containing(prepared) patient samples to be tested comprises, or is attached to,its own ejection head, and that the ejection head is able to be aimedtowards at least one microfluidic inline reaction channel(s).

Alternatively, in at least one other exemplary embodiment wherein the atleast one ejection head and at least one microfluidic inline reactionchannel are in a fixed position relative to each other, there can bemultiple ports leading to the microfluidic channel. In this exemplaryembodiment, each multiple ejection head is desirably aligned to one ofmultiple microfluidic ports (e.g., as many as there are ejection heads).Each microfluidic port leads to a single microfluidic inline reactionchannel (see, e.g., FIG. 2). In at least one further exemplaryembodiment, one ejection head can move between cartridges containing(prepared) samples to be tested (or blank cartridges for purposes ofcleaning the ejection head). After being attached to a cartridge, theejection head can be angled toward at least one microfluidic inlinereaction channel that is in a fixed position (or toward a waste capturemechanism when the attached cartridge is expelling waste (e.g.,conditioning buffer) or when the attached cartridge is a cleaningcartridge). In another exemplary embodiment, one or more ejection headscan remain in a fixed position, and the microfluidic chip can move suchthat at least one microfluidic inline reaction channel aligns withdifferent ejection heads at different positions.

In at least one exemplary embodiment of the invention, the cartridgecomprises an ejection head(s) (e.g., a printhead(s)) of the invention(see, e.g., FIG. 1A). In at least one other exemplary embodiment of theinvention, the cartridge, comprising the ejection head(s), is configuredto deliver a sample comprising genomic material to the microfluidicchip, e.g., to the microfluidic port(s) of a microfluidic inlinereaction channel(s) of the microfluidic chip. In at least one furtherexemplary embodiment, the sample comprising genomic material, as foundin, e.g., the printhead chamber E and/or the expelled (or ejected)sample (or sample droplets) directed to, e.g., the microfluidic port,further comprises reagent(s), e.g., reagent(s) inserted into thecartridge via a reagent insertion inlet 12.

2. Amplifying Genomic Material and Detecting Amplified Products

As described above, after a sample is prepared and sample droplets arerepeatedly ejected by a liquid ejection mechanism, the sample droplets,which can be approximately 1-25 pl (or some other appropriate volume)are analyzed with a microfluidic chip. In particular, a microfluidicinline reaction channel receives successive sample droplets of a sample(e.g., via a funnel and/or a microfluidic port), and sample droplets areanalyzed via automated methods. The total sample droplet volume (e.g.,the summation of ejected sample droplets as received together in, e.g.,the microfluidic port of a microfluidic inline reaction channel) canhave a volume in the range of about 3 picoliters to about 100nanoliters. Analysis comprises mixing with amplification and/ordetection reagents (in the reagent assembly area of the chip),amplifying the genomic material (in the amplification area of the chip)and detecting amplified products (in the detection area of the chip),and/or mapping the genomic material (in the matrix analysis area of thechip).

A. Microfluidic Port

Sample droplets can be ejected directly into a microfluidic inlinereaction channel. Additionally, it is within the scope of the inventionthat sample droplets be ejected into at least one microfluidic port,comprising an entry and a channel, and which leads to a microfluidicinline reaction channel (see, e.g., FIG. 2). In this exemplaryembodiment of the invention, a sample droplet can be ejected toward theentry of the microfluidic port, which is suitably sized to receive theejected sample droplet (e.g., is appreciably larger than the ejectedsample droplet), the microfluidic port channel, and/or the microfluidicreaction channel, etc., each of which can conveniently have a funnelshape. In at least one exemplary embodiment of the invention, a sampledroplet receiving system, e.g., a microfluidic inline reaction channeland/or a microfluidic port leading to a microfluidic inline reactionchannel, is configured to receive at least a portion of sample dropletscomprising genomic material ejected by an ejector head, e.g., an ejectorhead contained within a cartridge. In at least one other exemplaryembodiment of the invention, the entry of the port is ten times the sizeof a microfluidic port channel. In at least one further exemplaryembodiment of the invention, the widest diameter of the entry to themicrofluidic port is, e.g., 1 mm, and the diameter of the port channel,is, e.g., 100 μm.

Sample droplets can be transported through the port channel based on thenegative charge of the genomic material, i.e., electrokinetically,and/or with a pressure-driven flow. In at least one exemplary embodimentof the invention, the movement of a sample droplet within the portchannel is entirely, or in large part, controlled electrokinetically(e.g., by pairs of cathodes and anodes). For example, a cathode canconveniently be placed at the entry of each microfluidic port and ananode placed at the other terminus of each port channel (e.g., withinthe microfluidic inline reaction channel to which the port channel leads(see, e.g., FIG. 2)). To control the flow of fluid in each port if thereare multiple microfluidic ports, and consequently, multiple pairings ofcathodes and anodes, each pairing controlling the fluid movement withina particular microfluidic port can be activated only after theparticular microfluidic port has received one or more sample droplet(s)of a sample. It is also within the scope of this exemplary embodimentthat a sample droplet is transported into and through a microfluidicinline reaction channel via a pressure driven flow. Additionally,transport of a sample droplet through a port channel into a microfluidicinline reaction channel can be controlled via the use of well-known flowconstrictors.

Other well-known forces, e.g., surface tension, can be used to drive asample droplet through a microfluidic port, channel, and/or inlinereaction channel. For example, in at least one exemplary embodiment ofthe invention, a microfluidic port entry, channel, and/or inlinereaction channel can be filled with a buffer, e.g., the same buffer asthe eluate comprising genomic material, that promotes a collection ofsample droplets at the microfluidic port entry via, e.g., surfacetension forces. In this exemplary embodiment, it may be observed that asample droplet becomes part of the buffer, and does not remain adiscrete droplet. However, the electro-osmotic component ofelectrokinetic force creates a uniform pluglike flow of fluid down achannel, reducing or preventing diffusion of genomic material.Consequently, as used herein, sample droplet and sample plug (includingDNA sample droplet and DNA sample plug) refer to the plug-likecross-section of the continuous flow of fluid comprising the sampleejected as a sample droplet from the ejection head as the cross-sectionprogresses through a microfluidic port channel into a microfluidicinline reaction channel. Similarly, as described herein, a “primer plug”refers to the cross-section at any time of a continuous flow of liquidcomprising a particular “plug” of amplification and/or detectionreagents.

B. Microfluidic Inline Reaction Channel

Generally, as described above, a microfluidic inline reaction channelcan contain, e.g., a water-based liquid that provides the basis for thesample liquid. Alternatively, the microfluidic inline reaction channelliquid can be an organic-based liquid, for example, silicon oil of about60 poise. In at least one exemplary embodiment of the invention,repetitive sample droplets are ejected into a microfluidic inlinereaction channel and spacers separate the sample droplets. In at leastone other exemplary embodiment, air separates the sample droplets. In atleast one further exemplary embodiment, a hydrophobic substance, such asmineral oil, or some other organic-based liquid or solvent or the like,is used as a buffer spacer between each sample droplet in order tosurround and separate each sample droplet from the preceding orfollowing sample droplet in the microfluidic inline reaction channel. Inaddition, the inner wall of the microfluidic channels of a moleculardiagnostic device of at least one exemplary embodiment of the inventioncan be provided with a hydrophobic coating to decrease or preventcross-contamination between sample droplets. In at least one otherexemplary embodiment, as described above, the electro-osmotic componentof the electrokinetic force producing movement of the, e.g., sample plug(e.g., DNA sample plug) prevents or reduces diffusion of genomicmaterial in the plug. In other words, despite the movement inherent inmicrofluidics, the hydrophobic/hydrophilic difference between themicrofluidic inline reaction channel liquid and the buffer spacers(e.g., oil or air) enables a single DNA molecule to be kept in the plug(e.g., DNA sample plug) during its movement along a microfluidic inlinereaction channel without mixing with the buffer space, or with adjacentdroplets or plugs.

Generally, a microfluidic inline reaction channel of a microfluidic chipcan be 50 μm to 300 μm in diameter, and is typically 100 μm in diameter.As with the microfluidic port channel, a microfluidic inline reactionchannel can be a tube that is spherical, hemispherical, square, etc.,and formed in glass, quartz, plastic, etc. and can be formed ofdifferent materials depending on the area of the chip, e.g., can beformed with transparent material when it is within the detection area ofa molecular diagnostic device. Methods of forming microfluidic inlinereaction channels in a microfluidic chip are well known in the art. Amicrofluidic inline reaction channel can have any desired configuration,e.g., it can be straight, can form a joint or union with anothermicrofluidic inline reaction channel at a confluent junction, canseparate into two or more microfluidic inline reaction channels at aseparate junction, can allow the fluid within it to pool and/or mix,etc. Also, as described above, the flow within a microfluidic inlinereaction channel can be controlled by, e.g., electrokinetic forces,hydrodynamics (i.e., pressure), or a hybrid of the two.

In at least one exemplary embodiment of the invention, a “parent”microfluidic inline reaction channel can also be used for massive samplepartitioning. For example, a microfluidic inline reaction channel canlead into more than one “subchannels,” each of which can have, e.g.,one-tenth the flow rate of the parent microfluidic inline reactionchannel (see, e.g., FIG. 3). This structure facilitates the partitioningof each sample droplet into “subdroplets,” e.g., if a parentmicrofluidic inline reaction channel leads to ten subchannels, each withone-tenth the flow rate of the parent microfluidic inline reactionchannel, then each droplet will be divided into ten subdroplets, each inits own subchannel. This partitioning, especially when repeated, isreferred to herein as an embodiment of massive sample partitioning. Forpurposes herein, a microfluidic inline reaction channel encompasses suchsubchannels and a sample droplet encompasses such subdroplets.Additionally, the parent microfluidic inline reaction channel can betapered to effect the desired partitioning. Additionally, this method ofmassive sample partitioning can occur 1) prior to and/or after thereagent assembly area of the chip, 2) prior to and/or after theamplification area of the chip, 3) prior to and/or after the detectionarea of the chip, and/or 4) prior to or within the matrix analysis areaof the chip. A microfluidic inline reaction channel (or subchannel) canbe confluent with other microfluidic inline reaction channels, e.g., forthe addition of amplification and/or detection reagents, e.g., primerplugs.

C. Reagent Assembly/Amplification Area

FIG. 4 provides a nonlimiting example of a reagent assembly andamplification area of a microfluidic chip that comprises onemicrofluidic inline reaction channel. Of course, multiple microfluidicinline reaction channels and/or subchannels that run parallel to eachother are within the scope of the invention. In the microfluidic chip,each sample droplet (8) that is in a microfluidic inline reactionchannel (5) is further prepared at, e.g., a junction (10), e.g., aT-shaped junction, to form a sample plug by being mixed with a primerplug comprising amplification reagents (e.g., primer(s), nucleotides,polymerase, etc.) and optionally detection reagents (e.g., detectableagents, e.g., labels, fluorescent probes, intercalators, etc.). Askilled artisan will recognize which amplification reagents should bemixed with each sample droplet and at what concentrations the reagentsshould be used. For example, amplification reagents typically include apolymerase, dNTPs, magnesium, buffer, and a primer or a pair of primers.One of skill in the art will also be able to determine the primer orprimer pair to be used; e.g., if PCR is performed, a primer pair wouldbe appropriate. In contrast, if waveform-profiling analysis isperformed, a waveform primer would be appropriate. The design andselection of such primers are known in the art. Additionally, detectionreagents and methods of using such reagents to directly or indirectlylabel amplified DNA products are well known.

After a sample droplet has been ejected into a microfluidic port (ormicrofluidic inline reaction channel), and mixed with amplificationreagents to form a sample plug, it is transported along the microfluidicinline reaction channel into an amplification area of a device of atleast one exemplary embodiment of the invention, i.e., a firsttemperature-controlled area. As the terminology is utilized herein,similar to a sample droplet, a sample plug may or may not comprisegenomic material, and is considered a DNA sample plug if it doescomprise genomic material.

As sample plugs (which comprise sample droplets combined with primer(s),e.g., primer plugs) are continuously drawn along an inline microfluidicreaction channel (5), they are introduced to an amplification area,i.e., a first temperature-controlled area, such as a thermal controlplate (11). The path (12) of the microfluidic inline reaction channelcan be such that it facilitates the movement of each sample plug in awinding and reciprocated manner between low temperature areas (13) andhigh temperature areas (14) of the thermal control plate (11).

A skilled artisan will recognize that (A) the temperatures of the lowtemperature areas (13), the high temperature areas (14), and areasbetween the low and high temperature areas, (B) the path (12) of amicrofluidic inline reaction channel, and (C) the speed with which asample plug moves though a microfluidic inline reaction channel, can beappropriately adjusted according to the chosen amplification method. Forexample, the low temperature area (13) can be set to a temperatureappropriate to effectuate annealing and the high temperature area (14)can be set to a temperature to effectuate denaturing. Additionally, inat least one exemplary embodiment of the invention, the path (12) of amicrofluidic inline reaction channel is designed to facilitate themovement of a sample plug in a reciprocated manner between the lowtemperature and high temperature areas to effectuate, e.g.,approximately 20 to 40 cycles of denaturation, annealing, andelongation. Finally, the speed with which a sample plug (or DNA sampleplug) flows through a microfluidic inline reaction channel can be set toallow each sample plug (or DNA sample plug) to remain at a denaturing,annealing, or elongating temperature for an appropriate length of time.

As previously described, each microfluidic inline reaction channel, orportions thereof, can also be rapidly heated and cooled in a localizedand/or repeated manner such that the denaturing, annealing, andelongation steps of an amplification method (e.g., PCR, waveformprofiling), are executed as a sample plug moves along a microfluidicinline reaction channel and through a first temperature-controlled areaof a device of at least one exemplary embodiment of the invention. Forexample, Joule heating can be used to apply voltage to metal tracesalongside, inside, and/or crisscrossed with each microfluidic inlinereaction channel of a device of at least one exemplary embodiment of theinvention. Alternative methods of heating microfluidic inline reactionchannels include hot water, air, etc. Additionally, cooling of amicrofluidic inline reaction channel, or portions thereof, can beachieved through the use of cooling fluid that travels through a coil tocarry away thermal energy, or by facilitating rapid heat dissipation.Various methods of heating and cooling microfluidic inline reactionchannels and the like are well known.

The temperatures, the length of time at such temperatures, and thenumber of cycles to which a DNA sample plug are subjected vary asdesired to effectuate amplification of DNA for screening,identification, quantification, etc. For example, in at least oneexemplary embodiment, denaturing temperatures are between 90° C. and 95°C., annealing temperatures are between 55° C. and 65° C., and elongationtemperatures are dependent on the polymerase chosen (e.g., the optimalelongation temperature is about 72° C. for Taq polymerase). Also, theamplification method can comprise “hot starts” and/or a final incubationof a DNA sample plug at 75° C.

A sample plug can be moved through a microfluidic inline reactionchannel at different speeds ranging between about 50 μm per second andabout 5000 μm per second, e.g., about 500 μm per second. Varying thespeed with which a sample plug moves through a microfluidic inlinereaction channel can effectuate the duration of time a sample plugremains at a certain temperature (e.g., temperatures required fordenaturing, annealing, elongation, etc.) depending on the volume of thereaction, the concentration of the genomic DNA, etc. For example, atypical cycling profile is approximately 94° for 1 min., 60° for 1 min.,72° for 1 min. (a typical rule for a 72° C. elongation is 1 min for each1000 base pairs being amplified). In addition, the number ofamplification cycles required can determine the appropriate pathrequired of a microfluidic inline reaction channel.

After a sample droplet has been prepared, received by a microfluidicinline reaction channel, mixed with amplification reagents to formsample plugs, and the DNA within DNA sample plugs has been amplified,each sample plug is driven along the microfluidic inline reactionchannel into a detection area of the device, which can also be a secondtemperature-controlled area. As described above, a sample droplet canundergo massive sample partitioning in a microfluidic inline reactionchannel (i.e., in addition to massive sample partitioning via a liquidejection mechanism) at any time after being received by a microfluidicinline reaction channel and before being drawn into the detection areaof the microfluidic chip. A skilled artisan will recognize that only DNAsample plugs will comprise detectable amplified DNA products.

D. Detection Area

A molecular diagnostic device of at least one exemplary embodiment ofthe invention is designed to (A) allow DNA to be received as a sampledroplet(s) by a microfluidic inline reaction channel, (B) form sampleplugs in a reagent assembly area by mixing sample droplets with primerplugs comprising amplification reaction components and/or detectioncomponents, (C) effectuate the amplification of DNA as a DNA sample plugis advanced along the microfluidic inline reaction channel through anamplification area, i.e., a first temperature-controlled area, and (D)facilitate the detection of amplified DNA products as the DNA sampleplug passes through the detection area.

Passing a microfluidic inline reaction channel through a detection areawithin a second temperature-controlled area is within the scope of theinvention. Placement of a microfluidic inline reaction channel throughthe detection area within a second temperature-controlled area willsubject sample plugs traveling along the microfluidic inline reactionchannel to a temperature or temperature gradient (or sweep), i.e., oneor more temperatures, during detection. One of skill in the art willrecognize that detecting sample plugs as they are subject to atemperature sweep, e.g., detecting the fluorescence of a DNA sample plugat different temperatures, facilitates melting temperature analysis of,e.g., amplified DNA products.

FIG. 5 provides a nonlimiting example of a detection area of amicrofluidic chip of at least one exemplary embodiment of the invention.As a sample plug is drawn along a microfluidic inline reaction channel(5, as in FIG. 4) and exits the first temperature-controlled area (e.g.,11, as in FIG. 3), it proceeds downstream to areas related toidentification and/or analysis, e.g., it is introduced into a detectionarea, i.e., a second temperature-controlled area, which can be, e.g., asecond thermal control plate (16). A microfluidic inline reactionchannel can have a detection path (17) that facilitates the detection ofthe absence or the presence of amplified DNA in sample plugs, as thesample plugs move between lower temperature areas (18) and highertemperature areas (19). As sample plugs traverse through an opticalscanning area (20), any detectable reagent (e.g., fluorescent probes,intercalators, etc.) can be optically excited, e.g., with three-colorlaser beams, and any resulting emissions can be measured.

Generally, the lower temperature areas (18) of the detection area can beset to temperatures ranging between about 25° C. to about 65° C. Thehigher temperature areas (19) of the detection area can be set totemperatures ranging between about 55° C. to about 95° C. In the casethat PCR-amplified DNA is to be detected, the lower temperature areas(18) and higher temperature areas (19) of the detection area (16) can beset to one temperature, e.g., between about 25° C. to about 55° C.

The various instruments that can be used to regulate the temperatures inthe detection area, excite detectable reagents in DNA sample plugs, anddetect emissions, or a change in emissions, are commercially available.For example, temperature can be measured with, e.g., an infraredcharge-coupled device (CCD) (not shown) covering the optical scanningarea (20), or a larger or smaller scanning area. In at least oneexemplary embodiment, placement of precise temperature sensors on thesecond thermal control plate to calibrate the infrared CCD isrecommended to increase the accuracy of temperature measurements.

Subjecting a DNA sample plug to a temperature gradient or sweep in thedetection area enables detecting a waveform profile produced by waveformprofiling. As sample plugs traverse between temperatures, resultingemissions can be correlated with the temperature of a sample plug.Additionally, PCR-amplified DNA can be subjected to a temperaturegradient, although the emissions need only be detected at onetemperature. Alternatively, the lower temperature areas and highertemperature areas can be set to one temperature for the detection ofPCR-amplified DNA.

The detection stage optical system (not shown) can be used to detect thechange in emissions from amplified DNA, e.g., higher-order structures,as the amplified DNA is subject to a temperature sweep, by measuring,detecting, and determining the waveform profile of isolated DNA.Detection of a certain waveform profile can indicate that the screenedsample is contaminated (e.g., with bacteria), and subsequent comparisonof the resulting waveform profile with a database of waveform profilesproduced with a known primer(s) and DNA isolated from a knownorganism(s) can identify the contaminating organism. Additionally, ifisolated genomic material was concentrated within the sample liquid, andthe concentration known, the level of contamination can be quantifiedupon detection of the waveform profile.

A device of at least one exemplary embodiment of the invention can beeffectively utilized when little or no information is availableregarding whether a sample is contaminated and/or what organism iscontaminating a sample. Of course, the identity of an organism obtainedfrom a waveform profile can be further confirmed using the presentinvention to provide PCR product(s) for analysis. In at least oneexemplary embodiment of the invention, the identification of theorganism is further narrowed by forming several DNA sample droplets fromthe same organism, combining each DNA sample plug with a differentprimer chosen specifically to confirm the identity of an organism,amplifying each DNA sample droplet with a different primer (or set ofprimers) by, e.g., PCR or processes related to waveform profiling, anddetecting the absence or presence of amplified products. Correlating thepresence of amplified products with the particular primer(s) used canprovide the identity of the organism.

As described above, screening for the presence of an organism,identifying the organism, and/or quantifying the concentration of theorganism in a sample can be performed via waveform profiling and/or PCRusing a molecular diagnostic device of at least one exemplary embodimentof the invention comprising 1) at least one cartridge for extractinggenomic material, etc., 2) at least one sample droplet ejection head,wherein at least one cartridge comprises, or can be attached to, atleast one sample droplet ejection head for ejecting the genomicmaterial; and 3) at least one microfluidic chip for analyzing thegenomic material, wherein the microfluidic chip comprises at least onemicrofluidic inline reaction channel for receiving the ejected genomicmaterial from the sample droplet ejection head and at least one metaltrace or other component for heating of and/or fluid movement within themicrofluidic inline reaction channel, and wherein the at least onemicrofluidic inline reaction channel runs through a reagent assemblyarea, an amplification area within a first temperature-controlled areafor the amplification of DNA products, and a detection area. When a moredetailed examination of isolated genomic material is required, amolecular diagnostic device of at least one exemplary embodiment of theinvention can be used to select one or more DNA sample droplets, or DNAsample plugs (which may or may not have been amplified) from a sample ofinterest for mapping within the matrix analysis area of a microfluidicchip of at least one exemplary embodiment of the invention; in at leastone exemplary embodiment, the DNA in the sample droplet or plug has notbeen amplified. As described above, a sample droplet can undergo massivesample partitioning in a microfluidic inline reaction channel (e.g., inaddition to massive sample partitioning via a liquid ejection mechanism)at any time after being received by a microfluidic inline reactionchannel, including before or after being drawn into the matrix analysisarea of the chip.

3. Mapping of Genomic Material

The present invention can map the genomic information present in a DNAsample. Such mapping is contemplated to occur either (1) as a result ofa need or desire for more information in response to partialcharacterization of the contaminating organism (e.g., genus, species) byuse of the detecting steps outlined above, or (2) as a method ofcharacterizing contaminating organisms independent of utilization of thedetecting steps outlined above. Such mapping is further contemplated tooccur within the matrix analysis area of the chip of at least oneexemplary embodiment of the invention device. The mapping strategyimproves upon the recently reported technology known as direct linearanalysis (“DLA”; see Chan et al. (2004) “DNA mapping using microfluidicstretching and single-molecule detection of fluorescent site-specifictags” Genome Res. 14(6):1137-46, incorporated herein by reference in itsentirety).

In at least one exemplary embodiment of the invention, in response todetection of bacterial DNA (or some other form of genomic DNA that isbeing monitored in the detection area of the chip), a series of sampledroplets, e.g., a fresh series of sample droplets is produced by theliquid ejection head(s), as described above, and directed through atleast one microfluidic inline reaction channel leading to the matrixanalysis area of the chip. Dyes (intercalating and/or other dyes) areadded to the sample droplets to form sample plugs, either before orwithin the matrix analysis area.

The matrix is an integral part of the matrix analysis area of the chip.The matrix is formed on, and contained within, a glass substrate thatcontains several microfluidic inline reaction channels for movement ofmicrofluidic sample droplets through the units of the matrix analysisarea. Each unit (or “pixel”) of the matrix analysis area in turncomprises at least three components described herein. Each unit of thematrix analysis area is capable of (1) isolating and “stretching”molecules of DNA through the microchannel of a microfluidicDNA-stretching microchip; (2) generating beams of photons of visiblelight at more than one wavelength for exciting more than one dye boundto the molecules of DNA; and (3) detecting the results of theinteraction of the beams of photons with the dyes.

In at least one exemplary embodiment of the invention, each sampledroplet combines with a droplet (or plug) containing reagent(s)necessary for analysis. For this purpose a microfluidic inline reactionchannel can be confluent with other microfluidic inline reactionchannels, e.g., for the addition of the dyes and/or other reagentsnecessary for analysis in the matrix analysis area. In at least onefurther exemplary embodiment of the invention, the reagents include atleast two dyes that will bind to the DNA present in the droplets. One ofthe dyes is nonspecific and binds (or intercalates) with the DNA in anonspecific fashion (i.e., the DNA will be uniformly stained withintercalator) (see, e.g., Chan et al. (2004) Genome Res. 14(6):1137-46);the other dye is site-specific; for example, fluorescent peptide nucleicacids (PNAs) targeting, e.g., specific 7-8 bp sites (see, e.g., Chan etal. (2004)).

As described above (regarding the detection area of the chip), thedistribution of droplets to the several units of the matrix analysisarea can be accomplished by massive sample partitioning. The parentmicrofluidic inline reaction channel can be tapered to effect thedesired partitioning. For purposes of rapidly and evenly distributingthe sample droplets (and subdroplets) to the appropriate locationswithin the matrix analysis area, (1) the number of subchannels branchingfrom a parent channel may be any number that can be accommodated withina device of at least one exemplary embodiment of the invention (i.e.,10, 100, 250, 500, 1000, or more), and (2) subchannels can branch fromother subchannels. In at least one exemplary embodiment, subchannelsbranch from only other larger subchannels. In at least one furtherexemplary embodiment, 512 subchannels directly or indirectly branch fromeach of 512 larger subchannels, which in turn branch from a parentchannel(s). In addition, this massive sample partitioning can occurprior to or within the matrix detection area of the chip. A microfluidicinline reaction channel(s) or subchannel(s) can be confluent with othermicrofluidic inline reaction channel(s) or subchannels, e.g., for theaddition of dyes and/or other reagents necessary for the analysis stepsof the matrix analysis area by DLA technology.

In at least one exemplary embodiment of the invention, through a seriesof microfluidic inline reaction channels (parent channel(s), andsubchannels branching therefrom, and (optionally) smaller subchannels inturn branching therefrom), the matrix in the matrix analysis areacomprises a 512×512 matrix, therefore comprising approximately 260,000(e.g., 262,144) units (or pixels) for analysis of DNA molecules by theDLA technology. In at least one further exemplary embodiment of theinvention, the matrix can be 2×2, 3×3, 9×10, 10×10, 100×100, 256×256,1024×1024, or any appropriate square or nonsquare dimension.

The sample droplets or subdroplets can move through the channels andsubchannels of the device of at least one exemplary embodiment of theinvention by any number of means, and a predetermined rate(s) ofmovement of the droplets can be controlled by such means. For example,positive pressure applied to the beginning portion of a channel(s) orsubchannel(s) and/or negative pressure (i.e., a vacuum) applied to anend portion of a channel(s) or subchannel(s) or to a waste well or thelike can be utilized to move the droplets or subdroplets. In addition,electrokinetic forces can be used to move the droplets and subdroplets,as well as any other means known to one of skill in the art.

In at least one exemplary embodiment, the sample plugs, already combinedwith droplets containing reagent(s), proceed to a series of microfluidicinline reaction channels or subchannels that split off from the parentmicrofluidic inline reaction channel. A splitting device (“splitter”)controls the flow of droplets along the various microfluidic inlinereaction channels or subchannels in the series. For example, in anonlimiting example of a 3×3 matrix, three channels are able to receivesample droplets from the splitter.

In this exemplary embodiment, a series of droplets or plugs moves alongthe parent microfluidic inline reaction channel toward the splitter.Distal to the splitter are three channels (herein numbered 1, 2, and 3),each attached to a vacuum for pulling the droplets along themicrofluidic inline reaction channel. The first nine droplets (numbered1-9) are treated in the following fashion. The splitter allows dropletsto move into channel 1 (by means of the vacuum attached to channel 1)and droplets 1, 2 and 3 move along that channel until they are eachsituated in a predetermined location. The vacuum to channel 1 is thenreleased or removed, and then the vacuum cycle for channel 2 commences(i.e., the vacuum is applied to channel 2, moving droplets 4, 5, and 6into predetermined locations along channel 2). The procedure is thenrepeated for channel 3.

This procedure can be extended to any number of channels. In at leastone exemplary embodiment of the invention, 512 channels are utilized,and 512 droplets move along each channel until each droplet is situatedin a predetermined location. Thus a matrix alignment of 512×512 dropletsis present in the device at the end of the vacuum cycle for channel 512.

In the aforementioned 3×3 matrix exemplary embodiment, at the end of thevacuum cycle for channel 3, nine droplets are located in predeterminedlocations on the matrix. At each of these predetermined locations is anentry port to a “unit” (or “pixel”) of at least one exemplary embodimentof the invention. Thus, in a 3×3 matrix, there are nine (9) units of atleast one exemplary embodiment of the invention. A unit of at least oneexemplary embodiment of the invention, regardless of the number ofunits, comprises three main components. One component comprises a lightsource (e.g., a photon generator). A second component comprises amicrofluidic DNA-stretching microchip (see Chan et al. (2004)) orsimilar structure using direct linear analysis (DLA) technology. A thirdcomponent comprises a light detector (e.g., a photon detector).

Generally, there are several available forms of light sources anddetectors (i.e., active optics devices). In addition, there are severalavailable forms of passive optics, such as planar waveguides,microlenses, and filters, that can be used in combination with theactive optics. For reviews related to the use of various forms of opticsin microfluidic devices, see, e.g., Mogensen et al. (2004)Electrophoresis 25:3498-512; Sia and Whitesides (2003) Electrophoresis24:3563-76.

Among the light sources and detectors known to be useful in microfluidicdevices are light emitting diodes (LEDs), including organic lightemitting diodes (OLEDs) (e.g., the combinations of an LED with asingle-mode planar waveguide, a Si photodetector, and a microfluidicchannel cast in poly(dimethylsiloxane) (PDMS) is known in the art, as isthe integration of an LED, Si photodetectors, and microfluidic channelsby means of conventional complementary metal oxide silicon (CMOS)processing and sacrificial underetching). In at least one exemplaryembodiment of the invention, the light source (e.g., a photon generator)is also referred to as an emitter layer.

Lasers are also used in microfluidic devices. Vertical cavity surfaceemitting lasers (VCSELs) have been applied for near-infraredfluorescence detection of fluorophores spun onto a poly-(methylmethacrylate) (PMMA) substrate, in which the fabrication of thesubstrate also included a high pass filter and a photodiode fordetection. In at least one exemplary embodiment of the invention, afilter is also referred to as a filter layer, and a light detector(e.g., a photon detector, e.g., a photodiode) is also referred to as adetector layer.

Another useful light source is a microdischarge light source, e.g.,consisting of a metal anode and a microfluidic cathode filled with anaqueous solution of BaCl2 (which was used for excitation of DNAmolecules labeled with SYBR fluorophores).

Photodetectors for microfluidic systems (e.g., semiconductorphotodetectors) include systems in which the photodiodes or the likewere fabricated in the same substrate as a portion of the microfluidicchannels (e.g., in a device for DNA analysis, an interference filter canbe incorporated for suppression of excitation light). In anotherphotodetector system, a commercially available CMOS imager chip wasbonded to a microfluidic channel network cast in PDMS (measurements ofbromophenol blue and Orange G were possible, as was fluorescencemeasurements, because the CMOS imager incorporated an interferencefilter). Another known technique involves the use of low-temperaturethinfilm deposition techniques for production of amorphous siliconphotodiodes with filters on top of glass substrates (as opposed tointegration of the diode within a semiconductor wafer).

The integration of microlenses and planar waveguides in microfluidicdevices typically improves detection, e.g., by focusing the light in thechannel to increase the excitation power for fluorescence measurements,or by increasing the optical path length for absorbance detection usingplanar waveguides. A further advantage of planar waveguides is that beamsplitting can be accomplished for multi-point detection; thus verycompact devices can be realized when integrated with appropriate lightsources and photodetectors. Microlenses can be fabricated on top of thesubstrate (to shape the light in a path perpendicular to a wafer) orfabricated in the plane of the device. 2D planar microlenses can also beemployed in fabricating microfluidic devices. Planar waveguides (e.g.,polymer waveguides, glass waveguides, photonic bandgap sensors, etc.)can also be useful in designing microfluidic devices. Of particularinterest are photonic crystal structures: a photonic crystal can beviewed as the optical equivalent of a semiconductor crystal, and ischaracterized by a bandgap where light of a certain range of wavelengthsis not allowed to be transmitted. Further information regarding opticalsystems for microfluidic devices is available in the literature (e.g.,Mogensen et al., supra).

The light source (e.g., photon generator) and photodetector of at leastone exemplary embodiment of the invention, along with the DNA-stretchingmicrochip, are disclosed in more detail below.

A. Photon generator

The photon generator component (“PGC”), e.g., in the emitter layer,generates photons of in at least two different wavelengths (e.g., WV.Iand WV.II), and at least one of the wavelengths (e.g., WV.II) is splitinto two different beams (e.g., WV.IIa and WV.IIb) (see generally Chanet al. (2004)); several methods and devices to produce such photons insuch various wavelengths and beams are known in the art. The PGC isembedded in the glass substrate that comprises the matrix chip of atleast one exemplary embodiment of the invention, and a “light guide” canbe provided to direct the beams of photons toward at least two preciselocations in the microchannel of the microfluidic DNA-stretchingmicrochip (the “linear DNA microchannel”).

B. DNA-Stretching Microchip

In a microfluidic DNA-stretching microchip (“DNA stretchchip”),individual DNA molecules (e.g., double-stranded), bound with asequence-specific dye (or fluorescent tag) and with a nonspecific dye(or fluorescent tag), move through a “post field” and a “funnel,” and inthe process become linearized or stretched such that one DNA molecule ata time proceeds through a microchannel (the linear DNA microchannel) inwhich various beams and wavelengths of light are focused on the DNAmolecule. In at least one exemplary embodiment of the invention, thewidth of the linear DNA microchannel is 5 μm. In at least one otherexemplary embodiment of the invention, visible light is the form oflight.

A DNA stretchchip comprises a series of channels (e.g., a microfluidicinline reaction channel and/or subchannel comprising a post field and afunnel, and a linear DNA microchannel) etched by, e.g., photolithographyinto a substrate(s) and sandwiched between two substrates (e.g., asdetailed below).

As each molecule of DNA passes through the linear DNA microchannel, thestretched molecule encounters at least two sites at which beams ofphotons are focused. In at least one exemplary embodiment of theinvention, there are two sites at which the beams are focused: one site(the first site, as encountered by a molecule of DNA moving in thedirection of flow through the linear DNA microchannel) comprises twobeams of photons, one beam to detect the site-specific dye (e.g., WV.I)and one beam to detect the nonspecific dye (e.g., WV.IIa). At a secondsite, at some preset distance further along in the direction of flow inthe linear DNA microchannel, an additional beam of photons to detect thenonspecific dye (e.g., WV.IIb) is focused. The two beams detecting thenonspecific dye (e.g., WV.IIa and WV.IIb), which are set apart at somepreset distance, are used to establish the length of each molecule ofDNA as it passes through the linear DNA microchannel. The beam detectingthe site-specific dye is used to identify and analyze each molecule ofDNA. A site-specific dye that binds a relatively small site of severalbasepairs (bps) on a DNA molecule can be utilized for mapping (e.g.,identification and/or characterization) the organism from which suchgenomic DNA was extracted. As a nonlimiting example, the site-specificdye can target, e.g., a 7-8 bp site.

In some instances, the analysis of the positions of the site-specificdyes bound along the length of a double-stranded molecule of genomic DNAis known as “barcoding.” Such barcoding of the various molecules of DNAas they pass through the linear DNA microchannel can lead to theidentification of a particular type of genomic DNA (e.g., a taxonomictype, e.g., a barcode of a molecule of DNA can identify a particularfamily, genus, species, strain, etc. when compared with the barcodes ina database).

C. Photon Detector

In at least one exemplary embodiment, the photon detector (e.g.,photodetector, light detector, photon counter) component (“PDC”) of theinvention comprises at least one three-dimensional (3D) photonic crystalthat operates at a relatively low temperature and is formed or grown ona glass substrate, and at least one field emission transistor (FET),e.g., a thin-film transistor (TFT), that is also formed on a glasssubstrate (e.g., a TFT can have a thin film of silicon, and thetransistors are fabricated using this thin layer). In at least onefurther exemplary embodiment, suitable types of transistors other thanFETs can be employed. In at least one exemplary embodiment, the PDC isalso referred to as a detection layer. In at least one exemplaryembodiment, the photon detector is a digital photon detector. In atleast one other exemplary embodiment of the invention, glass is thesubstrate, although any other compatible substrates providing theoptical properties necessary for the present invention (e.g.,transparent plastics) are also contemplated. For example, several reviewarticles refer to multiple types of substrates useful in a chip of atleast one exemplary embodiment of the invention (see, e.g., Mogensen etal., supra; Sia and Whitesides, supra).

In at least one exemplary embodiment of the invention, photons from theseveral beams (e.g., WV.I; WV.IIa; WV.IIb) focusing on the DNA moleculespassing through the linear DNA microchannel are excited by the variousdyes (as explained above), and the excited fluorescent light isreflected to a “light guide” toward at least one photon detector. Thislight guideline inline detector(s) can be implemented with a photoniccrystal or crystals (e.g., for the three primary colors). In at leastone further exemplary embodiment, a 3D photonic crystal is capable offiltering so that more than one (e.g., two, three) wavelengths of lightcan reach the photon detector; in another exemplary embodiment, two,three or more different 3D photonic crystals (each filtering to allow adifferent wavelength) are employed. The photonic crystal(s) of at leastone exemplary embodiment of the invention is incorporated into each unitof the matrix chip; the photonic crystal(s) in the chip uses waveguidesto direct photons to the novel detector in the chip (inchip detector).The inchip detector is composed of a TFT photodetector. In someexemplary embodiments of the invention, multiple TFTs (e.g., two, threeor more TFTs) are incorporated into each unit of the matrix chip (tomonitor multiple wavelengths of light). In some further exemplaryembodiments, light amplifying TFT(s) are integrated into each unit ofthe matrix chip for efficient photon emission detection.

However, at least some TFTs, in standard form, are not well suited fordirect detection of photons of light. Therefore, in at least oneexemplary embodiment of the invention, a (pre-TFT) “gate” is constructedsuch that a photon of light energy is transduced to energy in the formof electrons that can be directly detected by the TFTs of at least oneexemplary embodiment of the invention. The gate comprises a gatingmaterial (“porphyrin gate material”), a major component of which is athree-dimensional (3D) single crystal polymer comprising porphyrins.This porphyrin gate material is excitable by photons, e.g., in thevisible spectrum, and can function as a semiconductor material. Fordisclosure related to this polymer and its use as a gate material, seeH. Segawa “Nanostructured molecular systems that freely manipulatephotons and electrons,” available atjstore.jst.go.jp/image/research/pdf/R99/R993100836.pdf; see also Segawaet al. (1994) J. Am. Chem. Soc. 116:11193-94; Susumu et al. (1995) Chem.Lett. (No. 10):929-30; Segawa et al. (1995) Synthetic Metals 71:2151-54;Susumu et al. (1995) J. Phys. Chem. 99:29-34; Susumu et al. (1995) J.Photochem. Photobiol. A: Chem. 92:39-46; Shimidzu et al. (1995) J.Photochem. Photobiol. A: Chem. 92:121-27; Susumu et al. (1996)Tetrahedron Lett. 37(46):8399-402; Shimidzu and Segawa (1996) Thin SolidFilms 273:14-19. Briefly, multiple porphyrins were linked (as porphyrinarrays) in a manner that created molecular systems that carried outphotoinduced electron transfer; these porphyrin arrays were directlylinked at the meso position. The porphyrin arrays were constructed inone-, two- and three-dimensional molecular architectures (see Segawa,supra).

The matrix analysis area can also comprise a CMOS-type gating mechanismand a CCD-type method of relaying the data from the several units of thematrix. Several methods for implementing such technologies to accomplishthe electrical data collection associated with the matrix device of atleast one exemplary embodiment of the invention are known in the art.

In at least one exemplary embodiment of the invention, the photondetector of the invention (as described above) reduces or eliminates theneed for an AC power supply (as is needed, along with an amplifier, forthe photon detectors described in Chan et al. (2004)). This improvedphoton detector of at least one exemplary embodiment of the inventionfacilitates considerable reductions in the power requirement (e.g.,through the use of TFTs) and the size of the instrument. This is anespecially advantageous consideration in light of the fact that eachunit (or pixel) of the matrix of at least one exemplary embodiment ofthe invention comprises a photon detector(s) associated therewith, and,in at least one further exemplary embodiment of the invention (i.e., a512×512 matrix), 262,144 units are incorporated into the matrix chip. Inaddition, the units of at least one exemplary embodiment of theinvention eliminate the need for external excitation lasers and therelated external optics systems disclosed in Chan et al. (2004).

The data produced by the detection of photons in the PDCs of the matrixanalysis area (i.e., in at least one exemplary embodiment, the detectionof electrons after transduction of the photon signal by theporphyrin-based crystal(s), e.g., porphyrin gate material, interfacedbetween the linear DNA microchannel and the TFT) is relayed to apredetermined destination, such as a recording device or other storagedevice, e.g., a computer chip or cache, etc., or a display device orother type of user-perceptible output interface. The coordination of therelay of data from the matrix analysis area to the predetermineddestination is accomplished by reading out data (representing detectedlight) at predetermined timings.

At least one exemplary embodiment of the invention is directed to amatrix configuration for reading out data representing detected light(i.e., in some exemplary embodiments, light transduced by the porphyringate material), as is depicted in, e.g., FIG. 6A. In at least onefurther exemplary embodiment of the invention, the matrix is a 512×512matrix of units (pixels), although for convenience only a two-by-two(2×2) matrix of units is shown in the figure.

The matrix comprises plural row wirings (RW), plural column wirings(CW), capacitors, phototransistors (e.g., the porphyrin gatematerial/TFTs described above), gating transistors, and binary shiftregisters (BSR1 and BSR2) that are interconnected in the manner shown inFIG. 6A. In at least one exemplary embodiment of the invention, thetransistors are FETs (e.g., TFTs), although in other exemplaryembodiments, other suitable types of transistors also can be employed.

Electrical signals representing light (e.g., light transduced by theporphyrin gate material) obtained in the above-described manner areapplied to the corresponding phototransistors, and, if the electricalsignal has a predetermined threshold voltage level, it is stored as acharge in the corresponding capacitor.

The manner in which signals are read out from the matrix can beunderstood in view of the timing charts shown in FIGS. 6A-6C. Forexample, application of a clock pulse (OUT 1 or OUT2; FIG. 6A) to thegates of the transistors connected to the corresponding row wiringresults in the charge being read from the capacitors. The clock pulses(OUT1, OUT2) are applied to those gates in accordance with Timing Chart2 (FIG. 6A), in response to a clock pulse generated by an overall systemcontroller (e.g., CPU) (not shown) being applied to the binary shiftregister (BSR1). The read out charge is then forwarded through thecorresponding transistor as a corresponding signal to the correspondingcolumn wiring connected to that transistor. The signal is then forwardedby the column wiring to the corresponding input (IN1 or IN2) of theassociated binary shift register (BSR2).

Timing Chart 1 (FIG. 6B) is an example representing how the measurementperiod (i.e., the period in which the photon is detected) relates to theperiod in which signals are read out from the capacitors, and the timingat which signals read to the row wirings corresponding to pulses OUT1and OUT2 are read within each read-out period. As shown in Timing Chart4 (FIG. 6B), signals are outputted from the capacitors coupled to therow wiring corresponding to clock pulse OUT1 within a first half of theread-out period, and signals are outputted from the capacitors coupledto the row wiring corresponding to clock pulse OUT2 within a second halfof the read-out period.

An example of the possible states corresponding to the signals appliedto terminals IN1 and IN2 of BSR2 (see FIG. 6A) is shown in Timing Chart3 (FIG. 6C). Data representing those states is outputted from the BSR2in series, according to the order in which it is received from thecolumn wirings.

The above-described timing charts, related to the matrix analysis area,comprise at least one exemplary embodiment that can be used inconjunction with the present invention. In other exemplary embodiments,other suitable timings can be employed, as long as the signals can beread out at distinct times for subsequent display, storage and/orprocessing.

A timing chart or charts can also be employed to control the movement ofthe sample droplets and subdroplets through the matrix analysis area,and to coordinate (1) the movement of subdroplets through the linear DNAmicrochannels and (2) the activation of the associated PGCs and PDCs ofthe units (or pixels) of the matrix analysis area.

The sample droplets or subdroplets pass through the DNA stretchchip at apredetermined rate, as taught generally by Chan et al. (2004).Essentially, the rate is to be set at a near-maximum rate that is stillaccurate for analysis of the dye-bound DNA molecules. One considerationis that the flow should not be at such a rapid rate that would allowmore than one DNA molecule to be present at the same time in the portionof the linear DNA microchannel in which the various beams of light arefocused. Another consideration is controlling the rate to avoidingclogging of the post field and the funnel elements of the DNAstretchchip. In at least one exemplary embodiment, a one-femtoliterdroplet (i.e., subdroplet, plug, etc.) traverses a DNA stretchchip of atleast one exemplary embodiment of the invention in approximately 0.12msec; in such an exemplary embodiment, the size of the matrix can beapproximately 512×512 units (or pixels). This exemplary embodiment iscalculated (1) to take into account appropriate levels of resolution,dynamic range, signal-to-noise ratio, etc. for the number of droplets(i.e., subdroplets, plugs, etc.) produced from, e.g., 2.5 μl of eluatecomprising genomic material (in turn, produced in the cartridge of atleast one exemplary embodiment of the invention from, e.g., 100 μl ofblood) and (2) for an approximate analysis time of one hour.

In at least one exemplary embodiment of the invention, the moleculardiagnostic device (comprising the cartridge, liquid ejection mechanism,amplification and detection areas, and matrix analysis area of at leastone exemplary embodiment of the invention) is portable (e.g., easilymovable, convenient for carrying). In at least one further exemplaryembodiment, the molecular diagnostic device is hand-held. In someexemplary embodiments, the device is useful to facilitate near-patient(i.e., on-site; away from a hospital or doctor's office) detection andanalysis of genomic DNA from, e.g., bacteria. In some further exemplaryembodiments, the device employs an AC power source and/or a DC powersource (see, e.g., the source voltage (Vs) in FIG. 6A). In at least oneother exemplary embodiment of the invention, the device functionswithout a need for an AC power source; e.g., the device functionsexclusively with DC power from an attached (or included), portable(e.g., hand-held) source.

In at least one exemplary embodiment of the invention, the device isexpected to produce mostly “zero detection,” i.e., most of the dataproduced by, e.g., the detection area of the device will show zerodetection of, e.g., bacterial genomic DNA. In this case, the detectionof, e.g., bacterial genomic DNA (i.e., “nonzero detection”) will lead toactivation of the matrix analysis area of the device and to droplet(i.e., subdroplet, plug, etc.) movement toward and into the matrix chip.In at least one further exemplary embodiment of the invention, suchactivation and movement in response to nonzero detection is automatic(i.e., is controlled by, e.g., a CPU).

As mentioned above, the barcode of a particular molecule of genomic DNAcan serve to identify the taxonomic category of the DNA (e.g., family,genus, species, strain, etc.). The barcode serves as a partialrepresentation of the nucleic acid sequence of the DNA, and can becompared to barcodes representing known taxonomic entities (e.g., in adatabase of know barcodes). The interpretation of the barcode data canbe automated and, in at least one exemplary embodiment, is included inthe molecular diagnostic device of the invention. In some exemplaryembodiments, the interpretation function is directly connected to thematrix analysis area of the device.

In at least one exemplary embodiment of the invention, theinterpretation function of the device is devised to account for theexpected variation in sequence that naturally occurs within a givenlevel of taxonomic identification (e.g., at the level of species). In atleast one exemplary embodiment, this expected variation can be up to a75% variation in sequence identity. In at least one further exemplaryembodiment of the invention, a probabilistic interpretation feature isincorporated into the interpretation function of the invention to aid inthe best-fit identification of the source of the genomic DNA beinganalyzed by the matrix chip of the device.

In at least one exemplary embodiment of the invention, a database (e.g.,comprising barcode information for known or expected pathogens) isincluded as part of the device. The barcodes of the unknown molecules ofgenomic DNA being analyzed in the matrix analysis area of the device canthus be compared and contrasted (e.g., matched) to the barcodes in thedatabase to potentially establish the identity of the unknown DNAmolecules (i.e., the DNA molecules being analyzed). The moleculardiagnostic device of at least one exemplary embodiment of the invention,comprising such a database, can then identify the source organismcorresponding to a given DNA molecule being analyzed and, through theinterpretation function comprising a probabilistic interpretationfeature, provide a calculation of similarity to various levels oftaxonomic identification (e.g., with an estimate of probable accuracy).For example, to the extent that the site-specific dyes and the barcodingof a particular exemplary embodiment of the invention are able toaccurately identify specific portions of the sequence of an unknownmolecule of genomic DNA, a set of barcode data for that molecule of DNAcan indicate 100% similarity or identity with a set of barcode data inthe database. In this instance, the device can identify, e.g. the genusand species of the source. In another instance, e.g., indicating 83%identity, the device can identify the same genus and species, but withan estimate of accuracy indicating the detected variation. In stillanother instance, e.g., indicating 50% identity, the device may onlyidentify the genus (with an estimate of accuracy for that level ofidentification) but give no indication as to species.

In at least one exemplary embodiment of the invention, the database isnot included as part of the device; rather the molecular diagnosticdevice is in wireless communication with a central database.

All scientific articles, reviews, patents, and patent applications citedin the present application are hereby incorporated by reference hereinin their entireties.

EXAMPLE

The Example which follows is set forth to aid in the understanding ofthe invention but is not intended to, and should not be construed to,limit its scope in any way. The Example does not include detaileddescriptions of conventional methods that would be well known to thoseof ordinary skill in the art.

Example 1 The Matrix Analysis Area of the Molecular Diagnostic Device

In at least one exemplary embodiment, the photon generator component(PGC) and the photon detector component (PGC) of a unit (of the matrixanalysis area of the molecular diagnostic device) are described as amicrocapillary waveguide with integrated optical filter, and depicted inFIGS. 7-11. A single wavelength optical filter is incorporated into halfof the microcapillary sandwich during manufacture. This results in theability to place the photodetector very close to the microcapillarydetection tube and thus increase the optical efficiency of receiving thefluorescence emission spectra. In terms of advantages regarding theexpense related to the device of at least one exemplary embodiment ofthe invention, fabrication costs can be much lower due to the fact thatthe microcapillary manufacturing material is shared with the opticalfiltering material (e.g., they are the same).

In at least one exemplary embodiment of the invention, the matrixanalysis area comprises an emitter layer, a filter layer, and a detectorlayer.

FIG. 7 shows a portion of one unit (or “pixel”) of the matrix analysisarea. As noted above, each unit has at least three components. In thisfigure, the PGC is the “photonic laser” and the PDC is the“photodetector.” The “capillary channel” (e.g., a semicircular capillarychannel) serves as a portion of the DNA stretchchip. Two plates of themicrocapillary device are shown in FIG. 7. One plate (lower) of themicrocapillary device, “dyed filter material and waveguide,” isfabricated from the desired colored filter material to control thespectral properties and wavelength(s) of fluorescence emission from thecapillary channel to the photodetector; in at least one exemplaryembodiment of the invention, the filter layer includes an optical filterdoped glass that passes a fluorescent wavelength and blocks the emitterwavelength. In at least one exemplary embodiment, the dimension d1 ofthe lower plate is in the range of about 10 microns to about 150microns. In at least one other exemplary embodiment, d1 is about 60microns. The other plate, an “excitation waveguide glass fluidic wafer”(upper plate in FIG. 7) is optically clear to allow the excitation light(e.g., emitter wavelength) from the laser to reach the reaction area. Amicrofluidic channel or subchannel (also referred to herein as acapillary channel) within a DNA stretchchip comprises, in at least onenonlimiting exemplary embodiment, an area (containing a post field) ofabout 50 microns in diameter or width, and a linear DNA microchannel ofabout 5 microns in diameter or width, with the fluid and DNA moving fromthe about 50-micron area to the about 5-micron DNA stretchchipmicrochannel through a funnel-like portion (e.g., reducing the width ofthe channel from about 50 μm to about 5 μm over a length ofapproximately 20 μm) (see, e.g., Chan et al. (2004)).

In at least one exemplary embodiment of the invention, the opticalfilter(s) and waveguide(s) used in the units of the matrix analysis areaand involved in the transmission of light (“laser excitation,”fluorescence) and “fluorescence emission” as shown in FIG. 8 cancomprise one or more optical materials, including but not limited tophotonic crystals, organically doped plastic resins, Schott opticalglass, colored quartz, colored glass, optical filter doped glass, etc.In at least one exemplary embodiment, the dimension d2 in FIG. 8 is inthe range of about 20 microns to about 300 microns. In at least oneother exemplary embodiment, d2 is about 120 microns. As stated above, inat least one exemplary embodiment, the filter layer includes an opticalfilter doped glass that passes a fluorescent wavelength and blocks theemitter wavelength. In at least one further exemplary embodiment, theunit comprises a 3D photonic crystal or crystals.

In at least one exemplary embodiment of the invention, units of thematrix analysis area, and specifically the microfluidic channelscontained therein, are manufactured by photolithography.Photolithography can be used to create the microfluidic channel(s),subchannel(s), and/or microchannel(s) in one side of a glass or quartzsubstrate (the photolithography process can be placed on either side ofthe substrate). The etched substrate is then bonded to another (e.g.,flat) substrate to form an array of microfluidic channel(s). A skilledartisan will recognize that channels of, e.g., semicircular shape incross-section are produced by bonding an etched substrate to a flatsubstrate, and that matching etching on both substrates prior to bondingcan be employed to produce a channel with a symmetrical shape incross-section (e.g., circular).

In at least one exemplary embodiment of the invention, multiple units,each comprising the three main components of a unit (i.e., PGC(s), DNAstretchchip(s), and PDC(s)), are matrixed together. A plurality of PDCscan be fabricated using a common integrated optical filter, as shown inFIG. 9 (see also a top view in FIG. 10). An advantage of this exemplaryembodiment is that one manufacturing piece can form the opticalfluorescence filter for many detection channels (i.e., linear DNAmicrochannels). In practice, when employing such a scheme, one mustconsider the possibility of optical “crosstalk” and employ methods toavoid this problem.

A skilled artisan will recognize the importance of the wavelengthcharacteristics and thickness of the glass filter(s) used in the matrixanalysis area of the chip. Examples of glass filter wavelengthcharacteristics are shown in FIG. 11. Using known methods in the art ofmicrolithography fabrication, optical glass filter materials can be inthe thickness range of 0.1 mm to 480 mm. In at least one exemplaryembodiment in the matrix analysis area, the thickness of the materialscan be in the range of 0.5 mm to 5 mm, or 1 mm to 3 mm. Using methodsand calculations well known in the art, a wavelength(s) andthickness(es) of filter material (e.g., in the filter layer) is selectedto separate the excitation spectra from the fluorescence emissionspectra with adequate optical SNR (signal-to-noise ratio); thus, thefilter layer can comprise a filter, e.g., an optical filter doped glass,that allows a fluorescent wavelength to pass and blocks the wavelengthof light from the emitter layer.

In at least one exemplary embodiment of the invention, multiplewavelengths of light (e.g., two, three, or more wavelengths) aremonitored by each unit (or pixel) of the matrix chip. In at least onefurther exemplary embodiment of the invention, multiple TFTs (e.g., two,three or more TFTs) are incorporated into each unit of the matrix chip(to monitor multiple wavelengths of light).

What is claimed is:
 1. A cartridge, configured to deliver a samplecomprising genomic material, comprising: (i) a genomic separation anddirection system, wherein the genomic separation and direction systemcomprises: a reaction chamber; and at least one binding and releasesubstrate, wherein the at least one binding and release substrate lieswithin the reaction chamber and is configured to bind and release atleast a portion of a sample comprising genomic material; (ii) an ejectorhead, wherein a first channel connects the reaction chamber and achamber in the ejector head, which ejector head chamber is configured toreceive at least a portion of a sample comprising genomic materialthrough the first channel from the genomic separation and directionsystem, and wherein the ejector head expels at least a portion of thereceived sample out of the cartridge as ejected sample droplets; and(iii) two electrodes, wherein a first electrode is positioned in thereaction chamber and a second electrode is positioned in the channelconnecting the reaction chamber and the ejector head.
 2. The cartridgeaccording to claim 1, wherein the substrate comprises charge switchmaterial.
 3. The cartridge according to claim 1, wherein the binding andreleasing of at least a portion of the sample is in response to anelectric voltage.
 4. The cartridge according to claim 1, wherein thebinding and releasing of at least a portion of the sample is in responseto a difference in ionic composition of a fluid in contact with thesubstrate, and wherein the sample is contained within the fluid.
 5. Thecartridge according to claim 1, wherein the binding and releasing of atleast a portion of the sample is in response to a difference in pH of afluid in contact with the substrate, and wherein the sample is containedwithin the fluid.
 6. The cartridge according to claim 1, wherein thesubstrate is a particle or bead.
 7. The cartridge according to claim 1,wherein the substrate is magnetic or paramagnetic.
 8. The cartridgeaccording to claim 1, wherein the substrate is bound to the innersurface of the reaction chamber.
 9. The cartridge according to claim 1,wherein the volume of at least a portion of the sample released from thesubstrate is reduced from the volume of the sample.